Université libre de Bruxelles
IRIBHM
A signalling function of
phosphatidylinositol 3,4-bisphosphate in
cell migration of breast cancer cells
Somadri Ghosh
Academic year: 2017-2018
Promotor: Prof. Christophe Erneux, PhD
Composition of the jury
President of the jury: Prof. Philippe Lebrun
External expert: Prof. Christophe AMPE
External expert: Prof. Jean-Baptiste Demoulin
Member: Prof. Nicolas BAEYENS
Member: Prof. Jean-Pierre BRION
Member: Prof. Pierre Roger
Acknowledgements
Firstly, I would like to express my sincere gratitude to my advisor Prof. Christophe Erneux for providing the continuous support of my Ph.D study and related research, for his patience, motivation, and immense knowledge. His guidance helped me in all the time of research and writing of this thesis.
I would like to thank the members of doctoral committee Prof Carine Truyens and Dr. Xavier De Deken for their insightful comments and encouragement. I would also like to thank Prof. Jean-Marie Vanderwinden for his support and suggestions throughout the thesis in regards to microscopic imaging and image analysis. I would like to offer my special thanks to Prof. Sabine Costagliola for giving me the opportunity to work in the microscope from her lab for live imaging. I would like to express my great appreciation to Dr. Isabelle Pirson and Dr. Benjamin Beck for their useful critiques on this research work. My grateful thanks are also extended to Dr. V. De Maertelaer for her help in doing the statistical data analysis, and all my collaborators who believed in me for the work.
I am particularly grateful to Televie and the Rose Foundation & Jean Hoguet funding to financially support the completion of my PhD thesis.
I would also like to extend my thanks to Ms. C Moreau, the technician of the laboratory for her kind help with various experiments during the project. I would like to thank my fellow lab mates with special mention of Dr. Elong Edimo, Dr. Anna Raquel Ramos, Dr. Sandra Koenig and Mr.Mathieu Antoine for their constant support during the research and for all the nice time spend over the last five years. Last but not the least, this PhD would not have been completed without the constant support provided by all the staffs and members of IRIBHM. My sincere thanks goes to Dr. Rashna Bhandari to introduce me to the field of phosphoinositols and phosphoinositides. I would also extend my thanks to all my friends for their support and understanding during the period.
I would also like to thank my parents, my wife and my family for their constant support and understanding especially during the hard times to pursue my dream of completing this Ph.D. thesis and life in general.
Table of Contents
Summary ... 1
Abbreviations ... 4
I. Introduction ... 7
1. Breast Cancer: ... 8
1.1 PI 3-kinase (PI3K) pathway and breast cancer: ...11
2. Phosphoinositides (PIs) ... 14 2.1 PI monophosphates ...16 2.1.1 PI(3)P ... 16 2.1.2 PI(4)P ... 18 2.1.3 PI(5)P ... 19 2.2 PI bisphosphates ...20 2.2.1 PI(4,5)P2 ... 20 2.2.2 PI(3,4)P2 ... 22 2.2.3 PI(3,5)P2 ... 23 2.3 PI trisphosphates ...24 PI(3,4,5)P3 ... 24 3. PI phosphatases: ... 25 3.1 PI 3-phosphatases: ...25
3.1.1 PTEN and PTEN related proteins: ... 26
3.1.2 MTM and MTM related proteins: ... 27
3.2 PI 4-phosphatases: ...28
3.2.1 INPP4A/B: ... 28
3.3 PI 5-phosphatases: ...29
3.3.1 Synaptojanin 1 and Synaptojanin 2... 30
3.3.2 OCRL-1... 31 3.3.3 INPP5B ... 32 3.3.4 INPP5J ... 32 3.3.5 SKIP ... 33 3.3.6 SHIP1/2 ... 33 3.3.7 Pharbin ... 34 4. SHIP2 ... 34
4.1 Structure and enzymatic activity: ...34
4.2 SHIP2 and insulin signalling ...36
4.3 SHIP2 interaction and cytoskeletal network ...36
4.4 SHIP2 inhibitors: ...39
4.5 SHIP2 and Disease:...40
4.5.1 SHIP2 in opsysmodysplasia:... 40
4.5.2 SHIP2 and breast cancer: ... 40
5. Phosphoinositides in cell migration and adhesion: ... 44
III. Results and Methods ... 51
Chapter I: SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration in 1321 N1 glioblastoma cells. ... 52
Chapter II: Fibroblasts derived from patients with opsismodysplasia display SHIP2-specific cell migration and adhesion defects. ... 77
Chapter III: Inhibition of SHIP2 activity inhibits cell migration, induces apoptosis and prevents metastasis in MDA-MB-231 breast cancer cells. ... 88
IV. Discussion and perspectives ... 140
1. Can the control of cell migration in 1321 N1 cells be generalized to other glioblastoma cells? ... 141
2. How does SHIP2 regulate cell motility in breast cancer cells? ... 142
3. Can we generalize the regulatory role of SHIP2 to non-cancerous cells? ... 144
4. Can we use AS1949490, a commercially available SHIP2 inhibitor, as a SHIP2 probe in cell migration? ... 145
5. SHIP2 and cell adhesion, how are they related? ... 146
6. Does SHIP2 affect the survival of breast cancer cells? ... 147
7. A new pathway to control PI(3,4)P2 via PTEN ... 148
8. Does SHIP2 play a role in tumour progression and metastasis of breast cancer cells? . 149 References ... 151
Summary
SHIP2 is a phosphatase that belongs to the family of the phosphoinositide 5-phosphatases. It is known to dephosphorylate PI(3,4,5)P3 to PI(3,4)P2 imparting a tight control of the PI 3-kinase pathway. Over the last decade, SHIP2 has been described as a tumor promotor or tumor suppressor in several cancer types such as glioblastoma, colorectal cancer or breast cancer cells. Several studies have proposed a role of SHIP2 in breast cancer cells, but its tumor promoting function was unclear at the beginning of this thesis especially in terms of its mode of regulation. In 2013, the INPPL1 gene that encodes SHIP2 has been found to be mutated in opsismodysplasia (OPS), a rare autosomal recessive disease characterized by delayed bone maturation but no molecular mechanism was provided to explain the mechanism.
In this thesis, we first contributed to establish a negative regulation of SHIP2 on cell migration in 1321 N1 glioblastoma (GBM) cells. Our studies revealed a dephosphorylation activity of SHIP2 on PI(4,5)P2 at the plasma membrane to control cell migration. This study was done in collaboration with Dr. Elong Edimo in the lab. We have also shown that the regulation of cell motility cannot be generalized to all the GBM cells. In LN229 and U-251 GBM cells we observed a positive regulation of cell migration by SHIP2.
We next took advantage of a unique model comparing fibroblasts derived from non-affected and OPS patients (in collaboration with Dr. Valérie Cormier-Daire). We have shown that the fibroblasts from the OPS patients are SHIP2 deficient and migrate slower as compared to fibroblasts from non-affected individuals.
Finally, the major part of the thesis was the study of breast cancer cells: in the model MDA-MB-231 cells, we established a positive regulation of SHIP2 on cell migration. We extended this regulation on cell migration to different breast cancer cell models using a SHIP2 inhibitor AS1949490. We confirmed that this inhibitor blocks the phosphatase activity of SHIP2 and showed its selectivity towards SHIP2 in cell migration assay. In MDA-MB-231 cells we deciphered a second messenger role of PI(3,4)P2 to control cell migration. Our data in this model rely on the use of SHIP2 depleted cells obtained by lentiviral infection and shRNA. We confirmed the positive role of SHIP2 on cell migration in the model of rat chondrosarcoma SHIP2CRISPR cells (in collaboration with Dr. Pavel Krejci).
A major goal of this thesis was achieved thanks to in-vivo studies: using MDA-MB-231 cells injected in SCID mice, we found a tumor promoting role of SHIP2 by determining the tumor weight. We also observed less lung metastasis of SHIP2 depleted injected cells as compared to control cells suggesting SHIP2 to be important for invasiveness of triple negative breast cancers.
Abbreviations
ARF ADP-ribosylation factor
ARP2/3 Actin related protein 2/3
BrdU 5-bromo-2’-deoxyuridine
BSA Bovine serum albumin
Btk Burton tyrosine kinase
C-terminal Carboxy terminal
DAG Diacylglycerol
ECM Extracellular matrix
EGF Epidermal growth factor
EGFR Epidermal growth factor receptor
ER Endoplasmic reticulum
Erk Extracellular signal-regulated kinase
ESRCT Endosomal sorting complexes required for transport FACS Fluorescence activated cell sorting
FAK Focal adhesion kinase
FCS Fetal calf serum
FN Fibronectin
GAPDH Glyseraldehyde-3-phosphate dehydrogenase
GBM Glioblastoma
GFP Green fluorescent protein
GOLPH3 Golgi phosphoprotein 3
HER2/ErbB2 EGF-like growth factor receptor
IHC Immunohistochemistry
INPP4A/B Inositol polyphosphate 4-phosphatase A/B
MAP Mitogen activated protein
MEF Mouse embryonic fibroblasts
MTM/MTMR Myotubularin/ Myotubularin related
mTOR Mammalian target of rapamycin
NHS Normal horse serum
N-terminal Amino terminal
N-WASP neuronal Wiskott-Aldrich Syndrome Protein OCRL Occuloceribrorenal lowe syndrome
OPS Opsysmodysplasia
PDK1 3-phosphoinositide dependent protein kinase-1
PH Pleckstrin homology
PI Phosphoinositide
PI(3)P Phosphatidylinositol 3-phosphate PI(3,4)P2 Phosphatidylinositol 3,4-bisphosphate PI(3,4,5)P3 Phosphatidylinositol 3,4,5-trisphosphate PI(3,5)P2 Phosphatidylinositol 3,5-bisphosphate PI(4)P Phosphatidylinositol 4-phosphate PI(4,5)P2 Phosphatidylinositol 4,5-bisphosphate PI(5)P Phosphatidylinositol 5-phosphate
PI3K PI 3-kinase
PKB/Akt Protein kinase B
PLC Phospholipase C
PR Progesterone receptor
PtdIns PhosphatydilInositol
PTEN Phosphatase and tensin homologue deleted on chromosome 10
SAM Sterile alpha motif
SH2 Src homology 2
SHIP SH2 domain containing inositol 5-phosphatase
SNX Sorting nexin
SYNJ1/2 Synaptojanin ½
TAPP Tandem-PH-domain containing protein
TGN Trans golgi network
WAVE WASP-family verprolin-homologous protein
WT Wild type
I. Introduction
1. Breast Cancer:
A major cause of morbidity and mortality in human is cancer, one of the main reason behind worldwide death costing up to 8.2 million lives in 2012 (Velloso et al., 2017). World Health Organization claims that breast cancer alone accounts for 25% of all cancer diagnosed in female worldwide (Velloso et al., 2017). Breast cancer is described to be clinically and morphologically a complex disease with heterogeneity arising from tumor size, age, histological grade and involvement of biomarkers. Last decades have focused on in-depth study for the molecular basis of this heterogeneous disease, deciphering cross-talk between several signalling pathways and effect of different treatments and responses of the patients, pointing out that personalized medicine could play a crucial role in the treatment of the disease (Eroles et al., 2012). Despite such advancement, breast cancer is the leading cause of cancer death in woman and constitutes 7% of all cancer death (Velloso et al., 2017). Recent reports suggest increased rate of breast cancer in the developed countries of North America, and West and North Europe. A report on cancer statistics published by the European Commission indicated93,500 deaths in 2014 from breast cancer with only 1000 men and all the majority women, counting up to 6.9% of all cancer death in woman and 15.5% of overall cancer death in the European Union (Cleries et al., 2017). In Belgium, 109 out of 100,000 women were diagnosed with breast cancer every year (Renard et al., 2011). The US reported 252,710 new cases and 40,610 deaths due to female breast cancer in 2017. About 20-30% of breast cancer patients develop metastatic tumors which till date remains incurable with an average survival of 2-4 years depending on the subtype (Eroles et al., 2012). Today treatment of metastasis and understanding the molecular mechanisms involving the development of new therapies has become the prime interest of research in breast cancer.
Immunohistochemical (IHC) detection of hallmark receptors has classically been treated as the way to classify breast cancer. Breast cancer is classified into two major groups of Luminal type, the major fraction, and the Basal type (Eroles et al., 2012). Luminal type is further sub-divided into Luminal A, Luminal B, and HER2 positive. Along with these, a new subtype of Claudin-low breast cancer has also been recently described in the literature (Figure 1).
Luminal A: It constitutes 50-60% of the total breast cancer and is the most common subtype characterized by expression of ER (Estrogen receptor), PR (Progesterone receptor) and typical absence of HER2/ErbB2 (EGF-like growth factor receptor) expression (Sorlie et al., 2001). This subtype has a low rate of proliferation, and so it also has a low expression of genes responsible for proliferation. Luminal A has a good prognosis and significantly low rate of relapse of 27.8%. This is reported to be the most infiltrating lobular carcinoma with an 18.7% incidence of bone metastasis and less than 10% metastasis in the lungs, liver, and central nervous system (Kennecke et al., 2010).
Luminal B: It constitutes 10-20% of the total breast cancer and has been described to have more aggressive phenotype than luminal A corresponding to higher proliferative rate and histological grade; it is characterized by a bad prognosis (Sorlie et al., 2001). The main difference between the two subtypes is the expression of proliferation related genes. Luminal B derived cells often also express EGFR and HER2. The most common site of metastasis is reported to be the bone leading up to 30% of all cases, and the second site is the liver accounting for 13.8% (Kennecke et al., 2010).
HER2 positive: Of the total breast cancers, only about 15-20% is HER2 positive; it is characterized by poor prognosis and overexpression of HER2 gene or other genes associated with HER2 pathway (Sorlie et al., 2001). They have also been reported to have an amplified expression of cell proliferation related genes thus making them highly proliferative. About Figure 1: (A) Mammary development linked with intrinsic subtypes of breast cancer
development. The figure depicts various subtypes of breast cancer classified according to the expression of the three key receptor signatures of breast cancer. Adapted from Prat et. Al, NatMed 2009. (B) Depiction of breast cancer subtype classification with respect to receptor expression. Adapted from Snadhu et.al, 2010.
A B
40% of HER2 positive cancers show mutations in the p53 gene. The IHC profile of this subtype is ER-/HER2 +, as a significant amount of tumors with IHC profile of ER+/HER2+ are grouped into the Luminal B subtype.
Basal-like: Representing only 10-20% of the total breast cancers have been named so, due to the expression of genes expressed in normal breast myoepithelial cells like P-cadherin, caveolin 1 and EGFR (Sorlie et al., 2001). They have been characterized by the absence of the three key receptors of breast cancer, ER, PR, and HER2; they are therefore also named as the group of triple negative. They have a prevalence of occurrences in women of African origin characterized by large tumor size, high mitotic index, and tumor necrosis (Bosch et al., 2010). This subtype has been described to have worse prognosis with high infiltrating ductal carcinoma. They have been reported to be the source of aggressive metastasis in the lungs, lymph nodes and central nervous system (Kennecke et al., 2010).
Claudin-low: This sub-type was recently identified in 2007 and represents only 12-14% of the total breast cancer (Herschkowitz et al., 2007). They are characterized by low expression of genes related to tight junction like Claudin-3,-4, -7, E-cadherin and ocludin. From immunohistochemical point of view, 80% of this subtype is triple negative and only 20% expresses the hormone receptors (Prat et al., 2010; Prat and Perou, 2011). They have been described to have poor prognosis with lower expression of cell proliferation related genes and high grade infiltrating ductal carcinoma. They also express a bundle of genes associated with epithelial to mesenchymal transition associating them with acquisition of cancer stem cell phenotypes (Hennessy et al., 2009).
The last decade has seen marvelous effort not only in classifying breast cancer into subtypes but also to determine the variability arising in various signalling pathways regulating cancer progression. One way of studying such pathways is provided by the use of established cell lines as model system. The first breast cancer cell line established in 1958 was BT-20, but the most popular and most widely used are the MD Anderson series of cell lines established in 1980’s. It is important to note that the breast cancer model in use corresponds to and reflects to which sub-type of breast cancer as described above. According to their genetic profiling the cells would certainly behave differently to different treatments (Holliday and Speirs, 2011). For example, the famous cell line of MCF7 belongs to the luminal A subtype, whereas MDA-MB-468 and MDA-MB-231 belong to the triple negative group of breast cancer
(Holliday and Speirs, 2011). Thus knowledge of such genetic and transcriptomic profiles of cell lines would make studies more effective with a high impact on improvement of outcome on breast cancer patient and will drift away from the “one marker, one cell line” studies in the past (Holliday and Speirs, 2011).
Targeting the pathways mostly altered having the potential to regulate cancer progression and metastasis is the way to go in the current era of cancer research. The major pathways reported to be altered in association of breast cancer are Estrogen receptor pathway, phosphoinositide 3-kinase (PI3K) pathway, MAP kinase pathway, JAK-STAT pathway, WNT pathway, TGFβ pathway and NFκB pathway (Velloso et al., 2017). For the purpose of this thesis, we will only discuss the alterations of the PI3K pathway in the next section.
1.1 PI 3-kinase (PI3K) pathway and breast cancer:
PI3K is a lipid kinase with the ability to phosphorylate the inositol head group of the phosphoinositides (PIs) (described in details in the next sections) at the D-3 position. According to their structure, mode of regulation and substrate specificity PI3Ks have been classified into three different classes (Table 1). Among these classes only class I PI3K consisting of class IA (PIK3CA, PIK3CB and PIK3CD) have been reported to have regulatory role in cancer (Lee et al., 2015). Upon activation of the heterodimeric class IA PI3K by external stimuli like G-protein coupled receptors or growth factor receptors, PI3K phosphorylates PI(4,5)P2 to generate PI(3,4,5)P3 triggering the signalling via activation of Akt/mTOR (mammalian target of rapamycin) pathway (Cantley, 2002). This signalling has been referred to play a central role in cell growth, proliferation, survival, motility and apoptosis and is under the tight control of different groups of phosphatases discussed in detail in the next sections of the thesis.
The PI3K/Akt/mTOR (PAM) pathway have been reported to be the most frequently altered pathway in human cancer and PIK3CA gene encoding p110α to be the most commonly mutated oncogene in breast cancer (Yuan and Cantley, 2008). The mutation is generally observed in 25% of breast cancer patients and reported to cause a gain of function leading to a constitutive activation of the PI3K/Akt pathway (Paplomata and O'Regan, 2014). The other mutations in the pathway frequently reported are in PTEN, about 67% basal-like and 22% in HER2 enriched tumors and Akt1 frequently in luminal tumors (Lee et al., 2015). Akt1 have
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Table 1: The Table summarizes various members of the PI3K family classified into different
classes. Regulatory and catalytic subunits are shown in the Table which forms heterodimers upon stimulation to convert the mentioned substrate to the specified product. Adapted from Thorpe et al., 2015.
been reported to inhibit cell migration and invasion by down regulating focal adhesion kinase (FAK) and β1 integrin whereas Akt2 promotes cell migration and invasion via F-actin and vimentin (Riggio et al., 2017). Thus alteration of Akt pathway may alter this important phenomenon dictating tumor progression under the action of PI3K directly or via its downstream target PDK1, which has also been reported to be amplified in 20% of breast tumors. These data make the members of the PAM pathway to be the potential targets for drug discovery.
Targeting the PI3K/Akt pathway in cancer has shown great potential over the last decades in
and has been reported to be selective to pan-class I PI3K and have no effect against class III PI3K or mTOR (Maira et al., 2012). A combination of PI3K inhibitor (BKM120) with the PARP inhibitor (olaparib) have been reported to work in synergy in-vivo to control tumor progression in mouse model prompting this combination to be an important approach to be studied in human clinical trial (Juvekar et al., 2012).
Figure 2: The PI3K pathway and inhibitors of the pathway tested in phase I-III clinical trials
on solid tumors and/or breast cancer treatment. Adapted from Lee et.al, 2015.
2. Phosphoinositides (PIs)
Myo-inositol, a cyclohexanol was first purified from leaves in 1887 by Maquenne (Irvine, 2016). The structure of myo-inositol is easily described by the Agranoff’s turtle whose body represents the inositol ring and the numbering proceeds from right front flippers and continues counter clockwise via the head to the other limbs (Figure 3 A/B). Esterification of this myo-inositol at the D-1 position connects the ring to a diacylglycerol (DAG) backbone via a phosphodiester bond to form phosphatidylinositol (PtdIns) (Figure 4A). Phosphorylated derivatives of PtdIns form the family of PIs which plays major role in cell signalling both in normal as well as in pathological conditions (Irvine, 2016). In naturally occurring PIs, the inositol ring can be phosphorylated at three different positions (3, 4, and -5 position) among -5 free –OH group of the ring. An orchestra of different PI-kinase’s and PI-phosphatases maintains the level and diversity of these signaling lipid molecules. Depending on the positions of the phosphate group, there are seven different members of the naturally occurring PIs, the three monophosphoinositides [ PI(3)P, PI(4)P, PI(5)P], three bisphosphoinositides [PI(4,5)P2 , PI(3,4)P2 and PI(3,5)P2] and one tris-phosphoinositide PI(3,4,5)P3 (Figure 4B) (Balla, 2013).
PtdIns is produced at the endoplasmic reticulum by esterification of DAG and myo-inositol, which is then distributed to different cellular locations. At the membrane, PtdIns under the action of different PI-kinases and phosphatases produces different PIs (Figure 5). Various studies have shown specific localization and pools of PIs to be important for cell dynamics. These pools of PIs are negatively controlled by the action of a phospholipase C (PLC)
A
B
Figure 3: (A Myo-inositol represented in the chair confirmation to depict steriochemically
correct position of the –OH groups. (B) Argnoff’s turtle representation of myo-inositol with the limbs demonstrating the orientation of the hydroxyl group on the inositol ring. Adapted from Irvine et al., 2016.
activity which breaks the phosphodiester bond releasing the DAG and the soluble inositol phosphate ring. This newly released ring of phosphorylated inositol under the action of different specific inositol kinases and phosphatases gives rise to the family of inositol polyphosphate, a significant group of important secondary messengers. They range from mono-phosphoinositol to poly-phosphoinositols which have been shown under various studies to have a tight control on cellular physiology. For the purpose of this thesis, we will not discuss soluble inositol family here.
The amount of PIs estimated in various cell of tissue types shows a huge variability ranging from 0.2-1% of the total cellular phospholipids content which was majorly detected to be PI(4)P and PI(4,5)P2. Although PIs are quantitatively minor phospholipid in the plasma membrane their activities in controlling cellular physiology have been shown to be very specific. PIs are synthesized at membranes where they interact with different protein molecules to generate or control a signal. The specificity of such interaction is fine-tuned by the ability of interaction of certain PIs to specific targets by virtue of certain domains such as the Pleckstrin Homology (PH) domain, GTP binding domain, or BAR domain (Balla, 2013). Emerging studies have shown the importance of such specificity in terms of different cytoplasmic and nuclear activities such as vesicular trafficking, autophagy, cell division, cell morphology, cellular migration and many more (Rudge and Wakelam, 2016). In the scope of this thesis, we highlight some of the most important features of each of the seven PIs, and focus more on their control in cancer metabolism.
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A
B
Figure 4: (A) Schematic representation of phosphoinositides and (B) Schematic structure of
2.1 PI monophosphates
The sub-family of PI-monophosphates has three members PI(3)P, PI(4)P and PI(5)P. Among these three PIs, PI(4)P contributes to 10% of the total PIs whereas PI(3)P and PI(5)P are present at comparable quantities (De Craene et al., 2017). The PI-monophosphates are generated either by phosphorylation of PtdIns or dephosphorylation of different PI-bisphosphatases.
2.1.1 PI(3)P
PI(3)P shows a universal occurrence throughout the evolution of eukaryotes predicting some important and conserved role of the lipid in regulating cellular function. PI(3)P is produced
Figure 5: A general view of inter-conversion of phosphoinositides under the action of kinases
(depicted by blue arrows) and phosphatases (depicted by green arrows). Adapted from Brown and Auger, 2011.
on the cytosolic side of the plasma membrane predominantly by the phosphorylation at the D-3 position of the PtdIns by the action of class II and class III PI3Ks (Schink et al., 2013). The turnover of PI(3)P is controlled mainly by the myotubularin/MTMR family of PI phosphatases (Figure 6). PI(3)P acts as crucial second messenger in activation of signal transduction from extracellular stimuli. It has also been shown to play a key role in endo or exocytic signal transduction into the cell from the plasma membrane or the other way around (Marat and Haucke, 2016). PI(3)P is the predominantly found in early endosomes and has been shown to affect endosomal fusion with the help of different effector proteins like small GTPase Rab5 (Ras-related protein), Phafin2 or Early Endosome Antigen-1 (EEA1) thus regulating endosomal membrane dynamics (Simonsen et al., 1998). This lipid is also known to interact with the sorting nexin (SNX) family of proteins that mediate recycling and retrograde signalling to the plasma membrane from the TGN. Specifically, interaction of PI(3)P with SNX3 has been shown to mediate retrograde transport to the TGN, thus playing a regulatory role in the Wingless/Wnt signalling (Xu et al., 2001).
Induction of autophagy in human cells leads to a complex formation on the ER membrane enriched with PI(3)P which interacts with DFCP1 (double FYVE domain containing protein 1) to translocate to the PI(3)P and autophagosomal proteins in order to initiate the formation of autophagosome.(Axe et al., 2008; Hamasaki et al., 2013). PI(3)P interacts with WIPI (WD-repeat protein interacting with phosphoinositides) proteins which in turn recruits further downstream proteins like Atg12, Atg5 and Atg16L important for autophagy (Polson et al., 2010). Mutations altering the interaction of PI(3)P with WIPI proteins are frequently observed in cancer and neurodegenerative diseases (Proikas-Cezanne et al., 2015). Such studies point out functions of different effector proteins of PI(3)P in endosomal dynamics, receptor sorting, and autophagy. This indicates the biological importance of this PI in both physiological and pathological conditions (Schink et al., 2013).
2.1.2 PI(4)P
PI 4-phosphate accounts for 45% of the total PIs in human cells (Payrastre et al., 2001). It is one of the very first PI identified by Jordi Folch in 1940’s from bovine brain in equal mixture with PI(4,5)P2. It shows enrichment in the Golgi complex and synthesized under the actions of PI 4-kinases like PI4Kα and PI4Kβ on their precursor molecule PtdIns (De Matteis et al., 2002; De Matteis et al., 2013). A fraction of PI(4)P is also produced by the action of different PI 5-Phosphatases like OCRL, SKIP, SHIP2 and synaptojanin 1/2 on PI(4,5)P2 and by the well-known PI 3-phosphatase PTEN on PI(3,4)P2 (De Craene et al., 2017).
PI(4)P has been shown to play pivotal role in vesicular protein cargo transport from Golgi to plasma membrane and endosomal compartments by influencing protein sorting into endosomes, protein transport from TGN to plasma membrane, changing the membrane
Figure 6: Localization of 3-phosphoinositides in the cells and the kinases and phosphatases
regulating them. Adapted from Marat and Haucke, 2016.
composition driving vesicle formation and localization of Golgi resident enzymes. PI(4)P forms a complex with FAPP1, FAPP2 (four-phosphate-adapter proteins 1 and 2) and ARF’s (ADP-ribosylation factors) at the TGN through their PH-domain and is involved in membrane sensing required for glycosphingolipid metabolism (Lenoir et al., 2015). PI(4)P rich TGN interacts with the protein GOLPH3 (Golgi phosphoprotein 3) which in turn binds to the motor protein Myo18 (myosin 18) to produce the tensile force at the membrane to efficiently form the tubule and vesicles (Dippold et al., 2009). Oxysterol binding protein (OSBP) interacts via its PH-domain with PI(4)P in the Golgi complex which then exchanges the PI(4)P with sterols between ER and Golgi (Perry and Ridgway, 2006). This lipid has also been recently reported to play a role in autophagy and lysosomal biogenesis (LeBlanc and McMaster, 2010; Sridhar et al., 2013). The large pool of PI(4)P at the plasma membrane along with PI(4,5)P2 has been shown to be important to recruit protein with polybasic motifs and to regulate ion channels (Hammond et al., 2012). Recent studies showed the tips of the primary cilia to be enriched with PI(4)P, instead of PI(4,5)P2 affecting the recruitment of TULP3 and thus altering the sonic hedgehog pathway (Chavez et al., 2015).
Recent reports suggested significant transcriptomic alteration of gene encoding PI(4)P metabolizing enzymes in different cancers or tumor cell lines. Specifically, studies have shown a huge increase in expression of PI 4kinaseβ (PI4KB) in breast cancer tumors and cell lines suggesting a link between PI(4)P metabolism alteration and malignancy. PI(4)P specific interactor GOLPH3 is frequently amplified in solid tumors and has been shown to result in oncogenic transformation and tumor proliferation via the mTOR dependent pathway (Scott et al., 2009). Thus the involvement of PI(4)P effector proteins in regulation of cancer cell survival and proliferation suggests this PI to be an amenable drug target for anti-cancer therapies.
2.1.3 PI(5)P
PI 5-phosphate is the most recently identified and least characterized PI in mammalian system. It compromises only up to 10% of the total PI monophosphates in eukaryotic cells at the basal level (Viaud et al., 2014). In human system PI(5)P is produced under the action of PIKfyve lipid kinase (phosphatidylinositol-3-phosphate 5-kinase type III ) on its precursor
PtdIns, although the major pool of PI(5)P is derived by dephosphorylation of PI(3,5)P2 by the MTM family of phosphatases.
2.2 PI bisphosphates
2.2.1 PI(4,5)P2
PI(4,5)P2 is the most abundant member of the PI family comprising up to 45% of the total PIs in mammalian cells and about 90% of the PI bisphosphate. It is predominantly present on the cytoplasmic side of the plasma membrane at a density of 4000 molecules/µm2 (Xu et al., 2003). Recent understanding of the field predicts three different enzymatic routes to be involved in PI(4,5)P2 metabolism in eukaryotes. The major bulk of PI(4,5)P2 is generated by PIP5K α, β, and γ, residing at the plasma membrane with PI(4)P as their preferred substrate (Clarke et al., 2008; Ishihara et al., 1998). Type I and type II PI4K has also been shown to produce PI(4,5)P2 from PI(5)P as a substrate at the Golgi complex (Lacalle et al., 2015). In mammalian system PI(3,4,5)P3 has also been shown to be dephosphorylated by the PI 3-phosphatases PTEN and TPIP (Transmebrane phosphatase with tensin homology and PTEN homologous inositol lipid phosphatase) to produce PI(4,5)P2. Thus exclusive localization of these enzymes creates separate and distinct pools of PI(4,5)P2 which can independently regulate different cellular signalling actions. The level of PI(4,5)P2 in the cellular compartment is controlled either by receptor activated enzymes like PLC, to generate DAG and IP3, or Class I PI3K to produce PI(3,4,5)P3 one of the crucial signalling molecule. PI(4,5)P2 is also dephosphorylated by PI 5-phosphatases or PI 4-phosphatases controlling the intensity of the signal trafficking in the cell (Figure 7A).
Major functions of PI(4,5)P2 at the plasma membrane have been attributed to its interactions with different proteins. A high number of adaptor and accessory proteins required for clathrin mediated endocytosis interacts with PI(4,5)P2 via their ENTH (Epsin N-terminal homology), ANTH (AP-180 N-terminal homology) or PH-domain. PI(4,5)P2 interacts with the ENTH domain of Epsin1 resulting in structural rearrangement to generate membrane curvature required for endocytic vesicle formation (De Craene et al., 2012; Itoh and Takenawa, 2002). PI(4,5)P2 is also known to interact with SNX5 and SNX9 that control cargo sorting and vesicle-endosomal fusion, which are known to be regulated by the PI 5-phosphatase OCRL-1 (Occulocerebrorenal Lowe syndrome protein) (Nandez et al., 2014). Interaction of this PI
with SNX5 in the early endosomes regulates EGF stimulation by recruitment of PI(4,5)P2 at the plasma membrane (Merino-Trigo et al., 2004) (Figure 7B). The head group of PI(4,5)P2 has been shown to interact with α-actinin, vinculin and profilin to regulate actin dynamics. PI(4,5)P2 has also been shown to regulate filopodia formation and promote cell migration in human osteosarcoma cells by its direct interaction with the proteins MIM (missing in metastasis) and IRSp53 (Insulin Receptor Substrate protein of 53 KD) (Mattila et al., 2007). Activity of small G-proteins like Rac, Rho, Cdc42, and Arf required for maintenance of actin dynamics has been shown to be regulated via PI(4,5)P2 (Santarius et al., 2006). PI(4,5)P2 has been also identified in the nucleus initially by immunological techniques (Martelli et al., 2011). Actually, the nuclear pool of PI(4,5)P2 has been suggested to be important to control various nuclear functions. PI(4,5)P2 has been shown to bind the transcription factor UBF (Upstream binding factor) to regulate transcription of rRNA. This PI in the nucleus may also regulate the poly(A) polymerase (star-PAP) activity to control expression of certain genes (Li et al., 2013; Mellman et al., 2008). PI(4,5)P2 have been reported to control ion-channel function either by producing DAG via PLC enzymatic activity resulting in activation of the channel or via direct interaction with channel proteins like KCNQ, Kir and transient receptor potential (TRP) channels (Hilgemann et al., 2001).
It is also shown in highly metastatic breast cancer cells, that the plasma membrane pool of PI(4,5)P2 is reduced via up-regulation of PLCβ1 and PTPRN2 (Sengelaub et al., 2016). This reduction in the plasma membrane pool of PI(4,5)P2 releases activated cofilin to the cytoplasm, which promotes cytoplasmic actin dynamics in-turn promoting cellular migration and metastasis.
2.2.2 PI(3,4)P2
PI 3,4-bisphosphate represents 10% of the total cellular PIs at basal state and is predominantly present in the plasma membrane. Class I PI3K activation via a variety of
Figure 7: (A) Schematic representation of enzymatic pathway of PI(4,5)P2 metabolism.
Adapted from Kolay et.al, 2016. (B) Sub-cellular distribution of PI(4,5)P2 in mammalian cell. PI(4,5)P2 is represented by orange stars, and the boxes show the PI(4,5)P2 effectors and phosphatases at the respective subcellular location. Adapted from Tan et. al, 2015.
A
B
extracellular stimuli leads to formation of PI(3,4,5)P3 which further leads to production of PI(3,4)P2 (Hawkins et al., 1992; Hawkins and Stephens, 2016). Activity of the PI 5-phosphatases to dephosphorylate PI(3,4,5)P3 at the D-5 position produces the major pool of PI(3,4)P2. Recent studies have shown the importance of PI 5-phosphatases in production site-specific pools of PI(3,4)P2 at early endosomes, dorsal ruffles and lamellipodia (Hawkins and Stephens, 2016). A recent study of clathrin mediated endocytosis have also suggested PI(3,4)P2 production by a direct kinetic activity of Class II PI3K, PI3KC2α, on PI(4)P (Posor et al., 2013). PI(3,4)P2 is largely metabolized under the influence of the PI 4-phosphatases INPP4A and INPP4B to produce PI(3)P (Hakim et al., 2012).
Effector proteins of PI(3,4)P2 like PDK1, Akt1/2, Arap3 are also known to bind PI(3,4,5)P3 via the PH-domains suggesting the signalling of PI(3,4)P2 to act alongside PI(3,4,5)P3 (Dowler et al., 2000). PI(3,4)P2 is linked to PI3K/Akt signalling which suggests regulatory role of PI(3,4)P2 in various biological processes like cell cycle, cell survival or glucose metabolism. The recent study also suggests an independent regulation of Akt2 by PI(3,4)P2 at the endosome (Li Chew et al., 2015). TAPP1 and 2 are the only proteins that specifically bind to PI(3,4)P2, and loss of this interaction in mice have been shown to enhance the insulin-dependent Akt activity in heart and muscle. This also promotes Akt activity in B-cell suggesting a feedback inhibition mechanism of Class I PI3K signalling by PI(3,4)P2 (Li and Marshall, 2015).
Recent studies in cancer cell models suggest various other effectors of PI(3,4)P2 which can regulate endocytosis as well as actin dynamics, which will be discussed in the next sections of this thesis.
2.2.3 PI(3,5)P2
PI 3,5-bisphosphate is the rarest form of PI constituting only up to 5% of the total PI pool in a eukaryotic cell with specific enrichment in the late endosomes (Takatori et al., 2016). It is proposed that direct phosphorylation of PI(3)P by the PI 5-kinase PIKfyve is responsible for synthesis of the major PI(3,5)P2 pool (Shisheva, 2008). The level of PI(3,5)P2 is maintained by the dephosphorylation activity of the PI 5-phosphatase Sac3 in mammalian cells.
The literature describes PI(3,5)P2 to have a regulatory role in range of various cellular functions such as endo-lysosomal morphology, trafficking, stress-induced signalling and
channel activity (Ho et al., 2012) (Figure 6). PI(3,5)P2 deficient cells in higher eukaryotes are characterized by dilated endo-lysosomes which have been predicted to originate from early and late endosomes and of lysosomes (Nicot et al., 2006). This predicts the regulatory role of PI(3,5)P2 in membrane fission and retrograde trafficking. PIKfyve binds to p40 and the kinesin adapter JPL to promote endosome to Golgi trafficking, further predicting a regulatory role of PI(3,5)P2 in membrane fission and cargo trafficking (Ikonomov et al., 2006). PI(3,5)P2 depleted mammalian cytosol have been reported to be impaired in multivesicular body formation required for sorting of the cargo (Sbrissa et al., 2007). The ESCRT-III subunit of the ESCRT complex (Endosomal sorting complexes required for transport) required for sorting has been reported to interact with PI(3,5)P2 thus exerting a regulatory role of this PI in multivesicular body sorting. PIKfyve knockout mouse has been reported to be lethal highlighting the importance of PI(3,5)P2 (Ikonomov et al., 2011). PI(3,5)P2 in the endosome releases cortactin from the actin network via direct interaction with actin-filament binding region of cortactin, thus regulating the membrane curvature and actin cytoskeleton (Henne et al., 2011). But till date only very few mechanistic regulation of this PI has been described in the literature and thus further studies are required for having an in-depth insight into the role of PI(3,5)P2.
2.3 PI trisphosphates
PI(3,4,5)P3
PI 3,4,5-trisphosphate is the most characterized PI in the family although it represents on 5% of the total cellular PI in eukaryotic cells (De Craene et al., 2017; Eramo and Mitchell, 2016). PI(3,4,5)P3 is almost undetectable in the cell at basal level but huge transient fluctuations have been reported after stimulation. Cellular stimulation from receptor protein kinases or G-protein couple receptors stimulates phosphorylation of PI(4,5)P2 by the Class I PI3Ks. PI3Ks phosphorylates the D-3 position of PI(4,5)P2 to transiently generate PI(3,4,5)P3 at the inner leaflet of the plasma membrane. The level of this transiently generated PI(3,4,5)P3 is tightly regulated by the tumor-suppressor PI 3-phosphatase PTEN to generate PI(4,5)P2. PI(3,4,5)P3 is also rapidly dephosphorylated by the enzymes belonging to the family of PI 5-phosphatase such as SHIP1/2, SKIP, INPP5J to generate PI(3,4)P2.
PI(3,4,5)P3 and PI(3,4)P2 have been reported to interact with a plethora of effector proteins of PI3K signalling through their respective PH-domains. Some of the important effectors being small GTPase proteins, PDK1, Akt, PLCγ which defines the importance of PI(3,4,5)P3 in cellular function of cell proliferation, apoptosis, cytoskeletal dynamics, cellular migration and cellular trafficking. Binding of both PI(3,4,5)P3 and PI(3,4)P2 to Akt PH-domain have been shown to be crucial for full activation of Akt and recruitment at the membrane and subsequent downstream pathway activation. Phosphorylation of Akt at Thr-308 by PDK1 and Ser-473 by mTORC2 leads to an allosteric activation of Akt. Activated Akt further prime phosphorylation of various downstream targets like Beclin1 (important for autophagy), p27 (important for proliferation), GSK3β (important for glycogen synthesis and cell cycle regulation) (Ma et al., 2008). PI(3,4,5)P3 also activates Cdc42 to mediate filamentous actin assembly thus promoting actin cytoskeleton rearrangement (Zheng et al., 2013). Dysregulation of PI3K signalling has been reported to be a leading factor in various cancer forms, diabetes, and cardiovascular and neurological diseases. A large number of inhibitors are commonly used to prevent the interaction between PIs and the Akt PH domain (Massacesi et al., 2016).
3. PI phosphatases:
A huge number of studies of a previous couple of decades have reported constitutive activation of the PI3K/Akt signalling due to mutation or amplification of the gene encoding PI3K or Akt in various human cancers. Along with it, various PI phosphatases functional alterations have also been reported to drive human cancer. PI phosphatases are able to dephosphorylate at position D-3, D-4 or D-5. In this section of the thesis we will discuss the properties of these phosphatases in pathology.
3.1 PI 3-phosphatases:
As the name suggests, this group of phosphatases have the ability to dephosphorylate the PI at the D-3 position and can be majorly divided into two distinct groups of enzymes 1) PTEN and PTEN related proteins and 2) MTM and MTM related protein (Figure 8).
3.1.1 PTEN and PTEN related proteins:
Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is the best known and most characterized PI phosphatase to play a role in human cancer. It was identified in 1997 by the group of T. Davis as a candidate tumor suppressor gene on chromosome 10q23 (Li et al., 1997; Steck et al., 1997). The most important and best described function of PTEN is to negatively regulate the PI3K signalling by dephosphorylating PI(3,4,5)P3 to produce PI(4,5)P2. Studies have shown high activity of PTEN for PI(3,4,5)P3 pointing out PTEN as an inhibitor of Class I PI3K (Maehama and Dixon, 1998). Germline G129E mutation of PTEN has been associated with Cowden disease which has been reported as a cancer predisposition syndrome (Hollander et al., 2011; Liaw et al., 1997). Mayers et al. showed that this germline mutation of PTEN has lipid phosphatase activity for PI(3,4,5)P3 pointing out the lipid phosphatase activity to be important in the tumor suppressor role of this gene (Myers et al., 1998).
In a tumor environment, PI3K signalling has been described to stimulate proliferation, protect from apoptosis, enhance cell migration and breaking tissue barriers hence promoting tumor metastasis (Rudge and Wakelam, 2016). PI(3,4,5)P3 have also been described to play a major role in angiogenesis. Thus, as a tight controller PTEN could be very well described as a tumor suppressor. This also explains different pathological conditions like Cowden disease, Bannayan-riley-ruvalcaba syndrome, Lhermitte duclos disease and proteus syndrome arising from germline mutation of PTEN to predispose to cancer (Di Cristofano and Pandolfi, 2000; Song et al., 2012). PTEN mutations have been reported in glioblastoma, prostate, kidney, melanoma, lungs, endometrial, bladder and breast cancer (Rudge and Wakelam, 2016). In most of these cases it has been reported as a mono allelic mutation in the early stages, whereas bi-allelic mutations are related to aggressive and metastatic cancer. In small cell lung cancer a mutational loss of protein phosphatase activity with intact lipid phosphatase active have been reported in a recent study. This mutation in the small cell lung carcinoma has suggested to alter binding of PTEN with other proteins thus altering the localization of PTEN thereby changing PI(3,4,5)P3 local concentrations (Tibarewal et al., 2012). PTEN has also been described to be extensively sensitive to oxidative stress, which inactivates its enzymatic activity.
The PTEN like phosphatase (PTPMT1) containing the PTEN active site have been reported to dephosphorylate PI(3,5)P2, PI(3,4)P2 and PI(5)P in-vitro (Rudge and Wakelam, 2016). Although there have been no reports this phosphatase to be mutated in any cancer tissue, it has been shown to be pro-apoptotic in cancer cells (Niemi et al., 2013).
3.1.2 MTM and MTM related proteins:
Myotubularin (MTM1) was identified in myotubular myopathy and have been shown to dephosphorylate PI(3)P and PI(3,5)P2 to produce PtdIns and PI(5)P, respectively, both in-vivo and in-vitro. MTMR proteins are homologs of MTM with similar phosphatase activity (Laporte et al., 1996). The family of this MTM proteins consists of 15 members, 9 out of which dephosphorylates PI(3)P and PI(3,4)P2 (Taylor et al., 2000a). Some of these proteins are able to dephosphorylate only one of the substrate and some of them can dephosphorylate both PIs. This substrate specificity of this family of proteins makes them important in regulation of physiological functions such as cell proliferation, and malignancy although no tumor tissue has yet been reported to have mutation in any of these proteins. MTM has been shown to control PI(3)P pool thus regulating cortical remodeling required for endosomal influx and integrin-mediated myofiber attachment (Ribeiro et al., 2011). A recent study suggested a reversible phosphorylation of MTMR2 at Ser58 to localize the phosphatase to early endosomes and to regulate PI(3)P level (Franklin et al., 2011). Mtmr2/Mtmr13 double knockout mice showed critical role of MTMR2 and MTMR13 in modulation of Akt-mediated signalling and thus in turn controlled growth /survival balance in cells and tissues (Berger et al., 2011). MTMR7 and MTMR2 have been pointed out to be anti-apoptotic via activation of caspase-9 (Hnia et al., 2012). MTMR1, MTMR2-4 which localizes at the late endosome has been suggested to control EGFR degradation and thus the EGF signalling (Tsujita et al., 2004).
3.2 PI 4-phosphatases:
Dephosphorylation at the D-4 position of PI and remove phosphate from PI(3,4)P2 and PI(4)P (Figure 9).
3.2.1 INPP4A/B:
INPP4A dephosphorylates PI(3,4)P2 at the D-4 position to produce PI(3)P. The C2 domain of INPP4A has been reported to directly bind PI(3,4)P2 and to regulate the catalytic activity (Ferron and Vacher, 2006). INPP4A overexpression in cells has been correlated with
Figure 8: The domain structure of PI 3-phosphatases. Adapted from Marat and Haucke, 2016
PTP domain: phosphatase domain; PEST: proline, glutamine, serine, threonine rich sequences; CC: coiled-coil domain; PDZ-B: PDZ domain binding sequence; GRAM: rab like GTPase activators and myotubularins domain; DENN: differentially expressed in normal and neoplastic cells domain; FYVE: Fab1, YOTB, Vac1 and EEA1.
reduction in PI(3,4)P2 levels in cells and hence reduction of Akt signalling. Loss of INPP4A in tumor xenografts has been reported to decrease apoptosis, promote cell proliferation and increase anchorage independent growth suggesting a tumor suppressor role like PTEN by regulating Akt (Ivetac et al., 2009).
INPP4B has similar substrate specificity as INPP4A and thus reduces PI(3,4)P2 in cells. Unlike INPP4A, INPP4B interacts with PI(3,4,5)P3 to regulate localization of the PIs in-vivo. Down-regulation of INPP4B in human mammary cells has been shown to enhance cell migration, anchorage independent growth and potentiate Akt signalling (Gewinner et al., 2009). Loss of INPP4B expression has been reported to be correlated with poor patient survival in breast cancer and ovarian cancer. Recent studies have shown decrease in INPP4B expression in human breast cancer cell lines with elevate Akt signalling and enhanced tumor formation, consistent with the frequent loss of INPP4B in human breast cancer (Fedele et al., 2010). Similar results have also been found in prostate cancer cell lines and patients with prostate cancer with low expression of INPP4B (Hodgson et al., 2011). These reports suggest a tumor suppressor role of both INPP4A and INPP4B by inhibition of PI3K/Akt signalling.
3.3 PI 5-phosphatases:
PI 5-phosphatases family of enzyme comprises of 10 mammalian members each of which having a catalytic domain of 300 amino acid length by virtue of which they gain the ability to dephosphorylate the D-5 position of PI(3,4,5)P3, PI(4,5)P2 and PI(3,5)P2 (Balla, 2013; Rudge and Wakelam, 2016). Only the first member of the family INPP5A also known as
Figure 9: The domain structure of PI 4-phosphatases. Adapted from Marat and Haucke, 2016.
PTP domain: phosphatase domain; PEST: proline, glutamine, serine, threonine rich sequences.
Type I inositol 5-phosphatase has no activity towards PIs and only dephosphorylate Ins(1,4,5)P3 and Ins(1,3,4,5)P4 at the D-5 position. Along with the catalytic domain the enzymes of the family also contains various functional domains like CAAX motif, SKICH, proline-rich, SAC and Rho-GAP (Figure 10). These domains form various complexes with specific adaptor proteins to localize them to specific cellular location conferring site-specific functions to the enzymes. PI 5-phosphatases have been often reported to be involved in the cellular processes of adhesion, migration, lamellipodia formation, cytoskeletal organization, as discussed below.
3.3.1 Synaptojanin 1 and Synaptojanin 2
Synaptojanin 1(SYNJ1) (INPP5G) and Synaptojanin 2(SYNJ2) (INPP5H) are two highly homologous 5-phosphatase enzymes having two PI phosphatase domain, the Sac1 like phosphatase domain and an inositol 5-phosphatase domain, have been reported to dephosphorylate both PI(4,5)P2 and PI(3,4,5)P3 as substrate. In-vitro studies show dephosphorylating activity of full-length SYNJ1 on PI(3)P3, PI(4)P due to its Sac1-like domain. SYNJ1 have been shown to be specifically expressed in brain and mutations have been reported in familiar forms of Parkinson’s disease, a neurodegenerative disease, whereas SYNJ2 is reported to be expressed in a wide range of tissues (Ramjaun and McPherson, 1996). Activities of SYNJ1 on this different pool of substrates have been reported to specifically regulating decoating of synaptic vesicles making it necessary for efficient synaptic vesicles recycling (Kim et al., 2002). SYNJ2 has been shown to play a critical role in formation of clathrin-coated pits (Rusk et al., 2003). SYNJ1 has also been reported to specifically interact with Rac1 (Malecz et al., 2000) suggesting a regulatory function in Rac1 mediated signalling. Down regulation of SYNJ2 in glioblastoma has been shown to inhibit cell migration, invasion and lamellipodia formation and to be regulated by Rac1 (Chuang et al., 2004). Recent studies have reported a copy number gain of SYNJ2 correlates with shorter survival and poor prognosis in breast cancer patients (Ben-Chetrit et al., 2015). This study revealed catalytic activity of SYNJ2 to be important for metastatic tumor spread and overall tumor growth in xenograft models thus suggesting an oncogenic role of the phosphatase.
3.3.2 OCRL-1
Mutation of OCRL (INPP5F) has been established to be the cause of X-linked human disease OCRL (Attree et al., 1992) characterized by congenital cataract, renal tubular acidosis, amino aciduria and mental retardation (Lowe et al., 1952). Some patients with the same mutation show milder symptoms limited to the kidney defect phenotype and are termed to be suffering from the Dent’s disease (Hoopes et al., 2005). Early studies showed a preference of OCRL for PI(4,5)P2 over PI(3,4,5)P3 as substrate (Zhang et al., 1995). OCRL was shown to be located in the Golgi and TGN which suggests a role of the phosphatase in maintaining the
PI(4,5)P2 pool for proper Golgi-originated trafficking in cells (Dressman et al., 2000). OCRL protein has been shown to associate with various clathrin-coated transport proteins further suggesting a role of OCRL trafficking between endosome and TGN (Choudhury et al., 2005). Studies have shown interaction of OCRL with Rab proteins and their effectors to recruit OCRL to the membrane (Hoopes et al., 2005). A study further showed interaction of OCRL with Rab35, suggested the phosphatase to be a key player in the last step of cytokinesis (Dambournet et al., 2011). Recent studies also indicated a role of OCRL in primary cillia development which needs to be further studied to understand the role of OCRL in the kidney pathology (Coon et al., 2012; Coon et al., 2009; Madhivanan et al., 2012).
3.3.3 INPP5B
INPP5B is highly similar to OCRL and has similar domain organization (Mitchell, 1989). Studies in cells suggested PI(4,5)P2 to be its substrate. INPP5B has been reported to have a wide tissue expression with specific enrichment in kidney, lungs, and testes (Speed et al., 1995). Early studies predicted a mitochondrial localization of the enzyme, but recent studies have shown its existence in Golgi and the ER (Williams et al., 2007). INPP5B knock-out mice have been reported to have normal appearance except having defective testicular function, characterized by defects in sperm motility, maturation and defects in sertoli cells (Hellsten et al., 2002; Hellsten et al., 2001). No human pathology has been directly attributed to INPP5B.
3.3.4 INPP5J
INPP5J also known as proline-rich inositol polyphosphate 5-phosphatase (PIPP), is widely expressed in various tissues and contains proline-rich sequences at both the C-terminal and the N-terminal end. It has been reported to dephosphorylate both PI(4,5)P2 and PI(3,4,5)P3 in-vitro, but in cells, it apparently only recognizes PI(3,4,5)P3 (Mochizuki and Takenawa, 1999). It is via PI(3,4,5)P3 hydrolyzing activity of the enzyme that PIPP is shown to control Akt activity and neurite outgrowth in PC12 cells (Ooms et al., 2006). PIPP interaction with its partners has also been reported to regulate nerve cell polarization, axon selection and neurite elongation (Astle et al., 2011). PIPP was reported to be frequently down regulated in
melanoma, and when overexpressed it reduces tumor size in xenograft model (Ye et al., 2013). Studies have correlated overexpression of PIPP with better prognosis of breast cancer (Takahashi et al., 2004).
3.3.5 SKIP
SKIP (INPP5K) is enriched in the skeletal muscle and kidney and has been reported to hydrolyze both PI(3,4,5)P3 and PI(4,5)P2 (Schmid et al., 2004). Structurally it contains two conserved domains, a PI 5-phosphatases domain and a SKICH domain at the C-terminal end responsible for translocation of the enzyme from Golgi/ER to the plasma membrane upon insulin stimulation (Gurung et al., 2003). Homozygous knock-out of SKIP is embryonic lethal and studies in heterozygous knockout mice showed an enhanced insulin in the skeletal muscle (Ijuin et al., 2008). A study in C2C12 myoblast cells showed a decreased expression of SKIP to elevate Akt activity in response to insulin (Ijuin et al., 2000). A recent study in PTEN-negative glioblastoma showed a higher expression and correlation with better survival of the patients (Davies et al., 2015). It is suggested that in PTEN-negative glioblastoma SKIP modulates cytoskeletal organization and migration by regulating intracellular level of integrin-induced PI(4,5)P2 (Davies et al., 2015).
3.3.6 SHIP1/2
SHIP1 (INPP5D) and SHIP2 (INPPL1) are the SH2-domain containing type III inositol 5-phosphatase. PI(3,4,5)P3 have been described in the literature as the main substrate for both enzymes (Blero et al., 2005; Pesesse et al., 1997) to generate PI(3,4)P2 and it is very important to note here that both the substrate and the product of this enzymatic reaction are required for complete activation of Akt (Franke et al., 1997). The major activation mechanism for SHIP1 and SHIP2 is via their recruitments to the plasma membrane. These proteins are generally a part of multi-protein complexes and perform tissue or cell specific functions under the regulation of such interactions (Batty et al., 2007). SHIP1 is specifically expressed in hematopoietic and spermatogenic cells whereas SHIP2 has a ubiquitous expression (Liu et al., 1998; Muraille et al., 1999). SHIP1 deficient mice have been reported to display myeloproliferative disease with resemblance to chronic myeloid leukemia
characterized by myeloid proliferation, splenomegaly, and vast myeloid infiltration of the lungs (Helgason et al., 1998; Liu et al., 1999). SHIP1 deficiency in mice has also been reported to cause B cell lymphoma (Hamilton et al., 2011). The SHIP1 knockout mice suffer from severe osteoporosis characterized by hyperactivation of osteoclasts (Takeshita et al., 2002). These mice also show a clotting defect due to platelet aggregation deficiency (Severin et al., 2007).
3.3.7 Pharbin
Pharbin (INPP5E) is the only Type IV inositol 5-phosphatase expressed in mammals, with specific enrichment in heart, testis and brain (Kong et al., 2000). This enzyme is located in the Golgi and plasma membrane and has been reported to be able to dephosphorylate both PI(3,4,5)P3 and PI(4,5)P2. Mutations leading to the loss of catalytic activity of pharbin have been reported to cause Joubert syndrome, characterized by midbrain-hindbrain malformation, retinodystrophy, nephronophthisis, liver cirrhosis, polydactyly and ciliopathies (Jacoby et al., 2009). Studies have shown localization of INPP5E in the primary cilium of the affected organs, and destabilization of the cilia due to the mutation in the enzyme. Depletion of INPP5E gene in mice has been reported to cause multi-organ failure associated with primary cilia defects (Bielas et al., 2009). INPP5E have been reported to be down regulated in metastatic adenocarcinoma as compared to primary tumors whereas an overexpression was reported in cervical cancer (Ramaswamy et al., 2003; Yoon et al., 2003).
4. SHIP2
4.1 Structure and enzymatic activity:
SHIP2, encoded by the gene INPPL1 on the human chromosome 11, is ubiquitously expressed and was cloned in 1997 (Pesesse et al., 1997). The protein SHIP2 contains several domains important for its function both including the catalytic activity as well as interaction with other proteins to form complexes to drive crucial intracellular activities (Figure 11). Residues 21-117 form the SH2 domain which is known to bind phosphotyrosine-containing peptide, residue 944-949 constitutes an SH3-binding motif and residue 983-986 constitutes an NPXY motif that can interact with phosphotyrosine binding (PTB) domains once tyrosine is phosphorylated, and C- and N- terminal proline-rich regions. Residue 1196-1258
constitutes a SAM (sterile alpha motif) domain, which has been reported to interact with a wide range of proteins like EphA2 receptor or PI3K effector proteins regulating Arf, Rho GTPase and Arap3 (Backers et al., 2003; Drayer et al., 1996; Raaijmakers et al., 2007; Zhuang et al., 2007). The catalytic activity of SHIP2 is conferred by the PI 5-phosphatase domain formed by the 314 residues from 419 to 732. SHIP2 shares a sequence similarity of 42% with SHIP1. Residue 725-863 have been associated to a C2 domain in SHIP1, and by comparing the sequence between SHIP1 and SHIP2, residue 742-884 have been suggested to be a C2 domain as well. (Thomas et al., 2017). The C2 domain of SHIP2 was recently crystallized and has been suggested to enhance the catalytic activity of SHIP2 in synergy with the phosphatase domain (Le Coq et al., 2017).
SHIP2 have been shown to be the very first enzyme to hydrolyze PI(3,4,5)P3 at the D-5 position to generate PI(3,4)P2 both in-vitro and in intact cells (Pesesse et al., 2001). Study on SHIP2 deficient MEFs reports an upregulation of PI(3,4,5)P3 in SHIP2-/- MEFs as compared to wild-type MEFs, and this is observed after short time stimulation by serum or H2O2 (Blero et al., 2005; Zhang et al., 2007). The data suggested that SHIP2 acts on agonist provoked cells whereas PTEN already acts already in unstimulated cells i.e. starved cells (Blero et al., 2007). Cells that are PTEN deficient have high levels of PI(3,4,5)P3. Due to its ability to dephosphorylate PI(3,4,5)P3 a modulator of Akt, SHIP2 have the potential and also have been shown to regulate many of the PI3K signalling. It is important to note here that both PI(3,4,5)P3 and PI(3,4)P2 can equally bind to Akt although this is still a controversial area (Franke et al., 1997). For example, it has been reported in mast cells from SHIP1-/- mice, that complete activation of Akt required both PI(3,4)P2 and PI(3,4,5)P3 (Scheid et al., 2002). Interestingly, in our lab it was reported that PI(3,4,5)P3 could be upregulated is SHIP2 depleted cells in response to serum or EGF with no modifications in the content of PI(3,4)P2 (Blero et al., 2007).
Figure 11: The domain structure of SHIP2.
B: Sequence of SHIP2:
C2 P
Classically, SHIP2 is expected to dephosphorylate PI(3,4,5)P3 at the membrane upon stimulation by serum, growth factor or insulin, but over the last decade SHIP2 in cellular extracts have been shown to recognize PI(4,5)P2 as well (Giuriato et al., 2002; Taylor et al., 2000b). It was reported that in SHIP2 deficient COS-7 cells there is an up regulation of PI(4,5)P2 as compared to the wild type cells. This also suggested that SHIP2 regulates PI(4,5)P2 and endocytic clathrin coated pit dynamics (Nakatsu et al., 2010). It has been shown in our lab that SHIP2 can be phosphorylated on S132 both in COS-7 cells overexpressing SHIP2 as well as at the endogenous level in human GBM 1321 N1 cells. It was reported that SHIP2 S132 translocate to the nucleus and controls cell cycle progression. In the nucleus, SHIP2 S132 is located in the nuclear speckles and demonstrate PI(4,5)P2 phosphatase activity (Elong Edimo et al., 2011; Elong Edimo et al., 2013). All this data suggest SHIP2 may control PI(4,5)P2 turnover both at the plasma membrane and in the nucleus.
4.2 SHIP2 and insulin signalling
Early studies on SHIP2 have reported SHIP2 as a key regulator of insulin signalling and a major role in diabetes. In 3T3-L1 preadipocytes and cerebellar granule cells overexpression of SHIP2 have been reported to attenuate PI3K signalling induced by insulin stimulation (Soeda et al., 2010; Wada et al., 2001).Transgenic mice overexpressing SHIP2 have been reported to exhibit signs of mild insulin resistance along with attenuated performance in test for memory, avoidance and recognition (Soeda et al., 2010), which has been related to metabolic syndromes and brain impairment. Metabolic syndrome like type 2 diabetes is a component of Alzheimer’s disease and a single nucleotide polymorphism of SHIP2 have been related to predisposition to Alzheimer’s disease (Accardi et al., 2012; Accardi et al., 2014). Mice expressing catalytically dead SHIP2 have been reported to have defects in muscle development, adipose tissue and female genital tract, with no alteration in glucose tolerance and insulin sensitivity (Dubois et al., 2012).
4.3 SHIP2 interaction and cytoskeletal network
The function and regulation of SHIP2 are not limited to its catalytic activity. SHIP2 through different interaction motifs binds various proteins to influence various processes like
cytoskeletal organization and function including apoptosis, endocytosis, proliferation, cell adhesion, and migration. In this section, we will discuss such control of SHIP2 in brief. Initial studies have suggested re-localization of SHIP1 to the actin cytoskeleton during platelet activation and aggregation (Giuriato et al., 2003). Similar re-localization of SHIP2 was also reported in platelets and even at basal level. The study suggested a higher affinity of SHIP2 towards the cytoskeleton when compared to SHIP1 (Giuriato et al., 2003). A study in poxvirus reported interaction between SHIP2 and actin tail helping the virus fusion to the plasma membrane (McNulty et al., 2011). This binding also requires phosphotyrosine, N-WASP (Neural Wiskott-Aldrich syndrome protein) and tyrosine kinases, and cells lacking SHIP2 have been reported to release less number of viruses (McNulty et al., 2011). This control suggests a significant role of SHIP2 in regulation the host virus interaction. The major product of SHIP2, PI(3,4)P2 has been shown to bind Dlg1, a key regulator of basolateral polarity (Awad et al., 2013). It has been reported in endothelial cells SHIP2 inhibition delocalizes Dlg1, Scribble, and β-catenin from cell-cell contact suggesting SHIP2 be important for localization of Dlg1 and thus maintenance of cell polarity (Awad et al., 2013).
SHIP2 have been reported to have a perinuclear cytosolic localization, and endogenous SHIP2 has also been found to localize to membrane ruffles (Thomas et al., 2017). Ruffles are membrane formation in moving cells formed by fresh polymerization of actin filaments. Immunoprecipitation study of ruffle from EGF stimulated COS-7 cells identified interaction between SHIP2 and filamin C (Dyson et al., 2001). The same study also reported a direct interaction between filamin A and B using a yeast two hybrid system. This study also suggested SHIP2 localization to the ruffles is via its direct interaction with filamin A, B, and C thus predicting a key regulatory role of SHIP2 in filamin mediated actin cytoskeleton organization. Immunoprecipitation of SHIP2 from the Triton soluble fraction of resting platelets identified a direct interaction between SHIP2, filamin, actin, and GPIb-IX-V to form a complex that regulates sub-membranous and cortical actin (Dyson et al., 2003).
Co-immunoprecipitation of SHIP2 in EGF stimulated COS-7 or C2C12 cells have identified a direct interaction with the scaffold protein intersectin 1 (Xie et al., 2008). It was further confirmed by the pull-down experiment in rat brain which also identified the C-terminal
proline-rich region to be the interacting domain between SHIP2 and intersectin. Intersectin 1 localizes SHIP2 to the clathrin coated pits early in pit formation (Nakatsu et al., 2010).
Directional migration is a crucial phenomenon for various cell types specifically during development, wound healing and metastasis. Communication between the extra cellular matrix and the cell during the process of cell adhesion and migration is communicated by the complex structure of focal adhesion which is often connected to actin fibers. SHIP2 has been shown to play regulatory role in cell spreading and cell adhesion the primary events that occur when a cell comes in contact with any extra cellular matrix. Immunoprecipitation studies in HeLa cells showed an interaction between SHIP2 and the adaptor protein p130cas. This adaptor protein belongs to the Cas family of adaptor proteins and has been identified to be a crucial regulator of actin organization and member of integrin meta-adhesome. Recent literature also suggests an important role of this adaptor protein in development and progression of various proteins. SHIP2 interacts with p130cas via the SH2 domain and localizes to the focal contacts and lamellipodia (Prasad et al., 2001). Transient overexpression of wild type SHIP2 in HeLa cells has been reported to adhere more as compared to cell over-expressing SHIP2 mutated in the catalytic domain or the SH2 domain suggesting a control of SHIP2 in regulation of cell adhesion and spreading. Yeast two hybrid system study suggested a direct interaction between SHIP2 and Vinexin (Paternotte et al., 2005). Vinexin is an adaptor protein established to control cell adhesion and cytoskeletal organization. This study also showed that SHIP2 interacts with Vinexin via its C-terminus and this interaction localizes SHIP2 at the cell periphery and enhances cell adhesion on collagen type I. Upon plating on Type I collagen, SHIP2 has been shown to be phosphorylated on tyrosine by Src kinase in the NPXY motif. Recruitment and activation of Src is established to be a crucial early process in cell adhesion. This study suggested a Src activity dependent association between SHIP2 and Shc protein, a control over cell attachment and spreading on collagen I (Prasad et al., 2002).
Single cell migration is regulated by a number of different networks of actin cytoskeleton. For this thesis we will consider lamellipodium, composed of branched actin network, and lamella, composed of more elusive organization of actin. A study in cultured podocytes described, activation of nephrin induces formation of a protein complex composed of SHIP2,
