GlialCAM is phosphorylated in primary rat astrocytes possibly by ERK2 and ASK1

In silico analysis predicts several potential phosphorylation sites in the GlialCAM intracellular domain and Moh et al. showed indirectly that the protein is phosphorylated, however, there was no direct evidence that the protein indeed is phosphorylated (Moh et al., 2005). In order to assess this point, we first determined whether we could immunoprecipitate GlialCAM using our anti-GlialCAM antibody in order to then isolate the protein from cell lysate treated with radioactive phosphate. Lysate from HEK293 cells overexpressing GlialCAM was incubated with the anti-GlialCAM antibody and the protein-antibody complex was precipitated using protein A/G coupled beads. Western blot analysis of the samples showed that GlialCAM can be immunoprecipitated using our antibody, while GlialCAM does not bind to the control antibody (Figure 26a).

Furthermore, no GlialCAM immunoreactivity was detected when incubating the GlialCAM or control antibody with lysate from normal HEK293 cells. GlialCAM was not depleted from the lysate with the protocol that was used as the protein could still be detected in the unbound fraction.

Once GlialCAM immunoprecipitation was established we pursued the phophorylation assay. GlialCAM overexpressing HEK293 cells and primary astrocytes were incubated with [³³P]-orthophosphate, the cells were lysed and GlialCAM was isolated by immunoprecipitation from the cell lysate. The samples were analysed by SDS-PAGE gel electrophoresis, transferred onto a nitrocellulose membrane and [³³P]-orthophosphate incorporation in GlialCAM was determined by autoradiography. As shown in figure 26b, [³³P]-orthophosphate could be detected when immunoprecipitating GlialCAM with the anti-GlialCAM antibody from GlialCAM overexpressing HEK293 cells and primary astrocytes. No radiation was detected when using a control antibody or when incubating lysate of normal HEK cells with the anti-GlialCAM antibody. The bands on the autoradiography correspond to GlialCAM, since a Western blot on the same membrane with anti-GlialCAM antibody detected the same bands (figure 26c). The additional bands detected on the Western blot correspond to the heavy chain of the antibodies used for the immunoprecipitation. Here we showed unambiguously for the first time that GlialCAM is phosphorylated and, interestingly, that it is phosphorylated in cultured primary rat astrocytes. Normalized to the protein amount as seen in the Western blot, the protein in the astrocytes appears to be highly glycosylated.

Figure 26. GlialCAM is phosphorylated in astrocytes and in HEK293 cells overexpressing the protein. A) For phosphorylation studies, we first determined that GlialCAM can be immunoprecipitated using the polyclonal anti-GlialCAM antibody. Lysate from HEK cells transfected with the empty plasmid (ctrl lysate) (1) or plasmid containing GlialCAM cDNA (2) was used for the immunoprecipitation (IP). IP with the anti-GlialCAM antibody results in a band corresponding to GlialCAM (arrow) when incubated with GlialCAM containing lysate (10) but not ctrl lysate (9). IP with an IgG isotype control with control (7) or GlialCAM containing (8) lysate did not render GlialCAM bands. Not all GlialCAM was IP:d as GlialCAM can be detected in the unbound fraction after IP with the GlialCAM antibody (6) and the IgG ctrl (5). No GlialCAM is detected in the unbound fraction

from the ctrl lysate (3 and 4). B and C) Normal and GlialCAM overexpressing HEK293 cells, and primary rat astrocytes where incubated with ³³P, lysed and GlialCAM was immunoprecipitated from the lysate using the anti-GlialCAM antibody. The samples were analysed by Western blot and exposed on an x-ray film (b) to determine ³³P incorporation into GlialCAM and analyzed by Western blot (c) with the anti-GlialCAM antibody.

A ³³P signal is detected in lysate of HEK293 cells overexpressing GlialCAM (left lane) and primary rat astrocytes (6th lane from the left). Control samples including HEK293 lysate and immunoprecipitation with a rabbit IgG isotype antibody do not show any ³³P radiation. The strong band at ca 50 kDa that is detected in all samples in the Western blot corresponds to the antibody heavy chain. The autoradiography was exposed overnight at -70ºC, while the Western blot was exposed for 20 seconds (an exposure time at which the radioactive signal is too weak to interfear with the Western blot results).

We wanted to go further and determine which kinases are involved in GlialCAM phosphorylation. For this purpose we cloned and purified the recombinant GlialCAM intracellular domain (GlialCAM-ICD). Originally, we cloned his tagged versions of GlialCAM-ICD with or without the transmembrane domain. However, we were not able to express these constructs in HEK293 cells (data not shown). Therefore, we designed a fusion protein containing the highly expressing ß2-microglobulin at the N-terminus followed by a caspase-8 cleavage site, 6-his tag and GlialCAM-ICD. The DNA encoding for the fusion protein was cloned by several consequtive PCR amplifications and transfered into the Gateway^TM technology vectors. A signal sequence for protein secretion was added to the 3’ end of the open reading frame. The protein was expressed in HEK/EBNA cells and purifed from the cell supernatant by metal affinity to Ni²⁺ NTA and size exclusion chromatography (figure 27). The purified fusion potein was cleaved using caspase-8, rendering two separate entities, namely ß2-microglobulin and his-tagged GlialCAM-ICD. The latter was purified again though metal affinity chromatography and separated from remaining uncleaved protein through cation exchange chromatography.

Figure 27. Purification of GlialCAM intracellular domain. a) The cDNA of a recombinant protein containing beta-2-microglobulin (B2MG), followed by a caspase-8 cleavage site (LETD), 6-his tag and GlialCAM intracellular domain (ICD) was cloned into the mammalian expression vector pEAK12d and expressed as an excreted protein in HEK293 cells. b) BenchMark protein standard (invitrogen) that was used for the gels stained with coomassie blue (c, d, e). c) The cell supernatant (CS) was passed through a Nickel column and the bound protein eluted with EDTA. Then, it was run through a size exclusion column (SX200) in order to remove contaminants. The fractions were analysed by SDS-PAGE and coomassie (left image) and by Western blot using an anti-B2MG antibody (right image). d) Fractions containing the protein were pooled together and cleaved with Caspase-8. The left most lane shows the uncleaved protein (lane 1), followed by the protein cleaved for 1 hour (lane 2) and for 2 hours (lane 3). Following cleavage the GlialCAM-ICD-6his residue was isolated using a Nickel column and the EDTA elution fractions containing the protein were pooled. e) To remove contaminants the solution (SM: strating material) was run through a SP sepharose cation exchange column and again the fraction containing the protein of interest (bracket) were pooled together. A G25 coarse size exclusion column was then used to change the protein buffer. f) The final solution was analysed by SDS-PAGE and Simply Blue staining under non-reduced conditions.

The purified his-tagged GlialCAM-ICD was used for in vitro kinase assays. The purified protein was incubated with different kinases, ERK2, JNK3, MEK, CK2a, ASK1 and PI3Kγ in the presence of radioactive [³³Pγ]-ATP. The samples were separated by SDS-PAGE gel electrophoresis and incorporation of radioactive phosphate was analysed by phosphoimaging and autoradiography. As shown in figure 28, a radioactive signal can be detected in the samples in which GlialCAM-ICD was incubated with ERK2 or ASK1, and the position of the band on the gel corresponds to the position previously observed for purified GlialCAM-ICD. MBP was used as a positive control for the assay and the protein is phosphorylated by all the kinases tested, although at varying levels.

Most of the kinases auto-phosphorylate rendering a signal that is also present in the negative control containing only the kinase. Whether GlialCAM is also a substrate for ERK2 and ASK1 in vivo remains to be determined.

Figure 28. The purified GlialCAM intracellular domain (GlialCAM-ICD) is phosphorylated by ERK2 and ASK1 in vitro. GlialCAM-ICD, MBP (ctrl pos) or Hepes buffer alone (ctrl neg) was incubated with different kinases in the presence of γ-³³P-ATP, the samples were separated on an SDS-PAGE gel and radioactivity was detected using a PhosphoImager. GlialCAM-ICD shows ³³P incorporation in the presence of ASK1 (arrow, right gel) and ERK2 (arrow, left gel), while MBP is phosphorylated by all the kinases tested and no signal is seen with buffer alone (except for kinase autophosphorylation). ERK2: Extracellular signal-regulated kinase 2; JNK3: c-Jun N-terminal kinase-3; MEK2: MAPK/ERK kinase 2; CK2a: Casein kinase II subunit alpha;

ASK1: Apoptosis Signal-regulating Kinase 1; PI3Kγ: Phosphoinositide-3 kinase gamma.

2.2.1.3.GlialCAM induces cell clustering while no cis homo