Protein phosphatases

Protein pho sphorylation is a powerfrrl p o sttranscriptional mechani sm whereby metabolic energy effects specific changes in protein function

(Alexander, 1990). For phosphorylation to function as a reversible signalling system, rapid and dynamic enzymes ^are required to dephosphorylate ttre target proteins and terminate the response. ^So the level of phosphorylation of any protein is balanced by the relative activities of protein ^kinases and phosphatases.

Recent discovery

of

^a

family

of transmembrane protein tyrosine phos-phatases has been reported. Their cytoplasmic domain is

highly

conserved

within

the members

of

the family.

In

contrast, the extracellular region is not

conserved,

in

order to create ligand specificity.

Little

is known about those puta-tive ligands. They are believed to transduce signals to the cell interior.

A

second class of protein phosphatases, specific

for

serine and threonine residues, is much better known (Cohen, 1989; Cohen and Cohen, 1989). ^These enzymes are involved

in

tumor suppression and cell division. They show broad and overlapping specificities. They were classified according their reactivity

with

activators and inhibitors. Four major classes were identified. Type 1

(PPl)

is inhibited by two thermostable

inhibitors (inhibitor

¹ and

inhibitor

2) and

activated by both

cAMP

and C&+ . Type 2 (PPz) is not affected by them. PP2 has been found to comprise three phosphatases (PP2A,PPZB and PP2C) ttrat can be distinguished by their requirement

for

cations. PP2B is activated by Ca2+ (via calmodulin) and is also called calcineurin (Armstrong 1989). PP2C is activated by

Mg2*. PPzAis

blocked by

low

amounts (1

nM)

of okadaic acid, a marine-sponge

toxin

^(Bialojan and Thkai, 1988), which is a powerful tumor promoter.

PPl

is blocked at higher concentration (15

nM).

This toxin is now used as a probe

in

the study of the regulation of ion channel by phosphorylation.

8. Secretion

Protein secretion from cells can take several forms

(Kelly,

1985; Whalley and Tatham, l99O; Rothman and

Orci,

L992

for

reviews). Secretion is

constitutive if

^proteins are secreted ^as fast as they are synthesized. There is no evidence that constitutive secretion (at least

in

lymphocytes) is modified by changing

lc&*li,but

^rather by altering the rate of protein synthesis (Tkrtakoff and Vassalli, 1978).

In

conffast,

if

the

newly

synthesized proteins destined

for

secretion are stored

in

secretory vesicles which are regulated to fuse

with

the plasma

membrane and release their hormones or neuroffansmitter upon stimulation by ^an appropriate agonist, the secretion is called

regulated.

Cells that regulate their secretion condense their secretory products, whereas those that secrete

constitutively do not.

Nascent proteins contain signals that determine their ultimate destination.

Membrane bound ribosomes synthesize tfuee major classes of proteins:

lysosomal proteins, secretory proteins and proteins spanning the plasma

membrane.

It

is possible, by labelling experiments, to show that secretedproteins are synthesized and glycosylated

in

the rough endoplasmic reticulum ^(RER).

Proteins that contain

in

their carboxy-terminal ^{part the sequence}

KDEL

(Lys-Asp-Glu-læu) are retained in the RER (Munro and Pelham, 1987). This sequence is necessary

for

the retention

of newly

synthesized proteins

in

the RER.

The other proteins pass

in

the

Golgi

complex, which is a stack

of

flattened membranous sacs where glycosylation events occur. The Golgi ^is the

granules, or the plasma membrane according to signals encoded by their three-dimensional sffuctures. The

Golgi

is differentiated

into

a cis compartment, a

medial

compartment and a

trans

compartment. Each part of the Golgi mediates different glycosylation functions. The proteins arrive

fromthe

RER to ttre cis part of the Golgi by transfer vesicles, which fuse

with it.

Proteins are then transferred by vesicles budding and fusion

from

one compartment of the Golgi to another.

Membrane asymmeffy is preserved

in

these ffansport processes. From the ffans-Golgi,

newly

synthesized vesicles travel to the cell surface. The newly synthesized proteins reach the cell surface minutes after leaving the Golgi.

Regulated secretory cells are able to release,

for

a short period, large amounts of proteins. This is achieved, ^as mentioned previously, by storing newly synthesized proteins

in

secretory vesicles that have ^a

half-life

of days.

In contrast

to the

transport

vesicles of the

constitutive proteins,

secretory vesicles (where the

material

is condensed)

for

the regulated

proteins

are prevented

to

fuse

with

the plasma membrane,

until

the level of a second messenger,

usually lC**liris

^{raised. GTP seems also to play a role,}

^by

activating a myriad

of

small ras-related G proteins ^(I\d, around 20 Kd) which are

important

for

the different translocation steps that occur (Rothman and Orci, 1992). Some laboratories have now shown that trimeric G proteins (specially

G3)

are also involved

in

the conffol of vesicular traffrc through the Golgi complex (Burgoyne, 1992).

Another important point to mention here is that some cells like mast cells or neutrophils secrete the content of their secretory vesicle toward any part of the plasma membrane that is stimulated. They are called

non-polarized

secretory

cells.

In

conffast, epithelial cells

like

liver, endocrine or exocrine cells secrete their ^products

from

a small part of their plasma membrane. They are called

polarized

cells.

Part

of

our

work

during this thesis was based on the development of a simple new technique

for

the rapid monitoring of both

[Cf*]i

^{raises and}

exocytosis at the single cell level. This is reported in publication

tr.

In

pituitary

cells in primary culture, PRL is stored into dense core membrane bound secretory granules or

in

small vesicles. Both organelles may serve the release of PRL

via

exocytosis. The diameter and the number

of

secretory granules vary among PRL cells (e.g.

Txier -Vidal

et al., 1988).

An

exfteme situation is represented by GH3B6 and GHaCI tumor cells which store very

little

PRL

in

a few small secretory granules (publication

Itr).

Dans le document Control by protein phosphorylation and G proteins of calcium oscillations, and pituitary hormone secretion (Page 61-65)