The 93 kDa protein gephyrin and tubulin associated with the inhibitory glycine receptor are phosphorylated by an endogenous protein kinase

Abstract

The 93 kDa protein gephyrin is a tubulin binding peripheral mcmbranc protein that is associated with the inhibitory glycinc receptor and has been implicated in its anchoring at central synapses. Here, we demonstrate that gephyrin as well as co-purifying tubulin are phosphorylated by a kinase aclivity which is cndogenous to highly purified glycine receptor preparations. This kinase phosphorylates scrine and threonine residues and utilizes ATP, but not GTP, as phosphate donor. Its activity is not affected by various activators and/or inhibitors of cyclic nucleotidedependent kinases, calcium/calmodulin-dependent kinascs, or protein kinase C. A five-fold stimulation of kinase activity was, however, observed in the presence of poly-lysine. Phosphorylation of gephyrin and/or tubulin might regulate receptor/cytoskeleton interactions at postsynaptic membrane specializations.

X-ray Crystal Structure of the Trimeric N-terminal Domain of Gephyrin

“…Presently, mutants of the trimer interface, which would generate monomeric gephyrin or E. coli MogA, are not available to test the role of oligomerization for enzymatic function. However, oligomerization of native gephyrin has been observed previously in sedimentation studies (45) and suggested to play a role in the clustering of inhibitory receptors at developing postsynaptic membrane specializations (15).…”

Gephyrin: a central GABAergic synapse organizer

“…However, gephyrin has been identified as a phosphoprotein when copurified with the GlyR (which has kinase activity), 40 and mass spectrometric analyses of rat and mouse brains revealed that gephyrin has 22 phosphorylation sites, most of which are located within the C domain (except for threonine 324, which lies in the E domain 25,[41][42][43] ) (Figure 1). These modifications might induce conformational changes by affecting the structure of the C domain or the neighboring G and E domains, thereby altering the clustering, trafficking and binding properties of gephyrin.…”

Phosphorylation of Gephyrin in Hippocampal Neurons by Cyclin-dependent Kinase CDK5 at Ser-270 Is Dependent on Collybistin

“…Earlier studies reported that gephyrin in GlyR preparations from rat spinal cord is phosphorylated (22). Therefore, we used phosphatase treatment to remove all phosphates from gephyrin and compared the binding of mAb7a and Ab-175 antibodies in immunoblots.…”

“…Phosphorylation of gephyrin was first reported in 1992 (22). More recently, the binding of gephyrin to the peptidyl-prolyl cis/trans-isomerase Pin1 was analyzed and allowed the identification of amino acid residues (Ser-188, in the C-domain as major determinants of phosphorylation dependent Pin1 binding and also of gephyrin clustering (23).…”