title: Phosphorylation of synaptic GTPase-activating protein (synGAP) by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (CDK5) alters the ratio of its GAP activity toward Ras and Rap GTPases.
abstract: synGAP is a neuron-specific Ras and Rap GTPase-activating protein (GAP) found in high concentrations in the postsynaptic density (PSD) fraction from the mammalian forebrain. We have previously shown that, in situ in the PSD fraction or in recombinant form in Sf9 cell membranes, synGAP is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), another prominent component of the PSD. Here, we show that recombinant synGAP (r-synGAP), lacking 102 residues at the N terminus, can be purified in soluble form and is phosphorylated by cyclin-dependent kinase 5 (CDK5) as well as by CaMKII. Phosphorylation of r-synGAP by CaMKII increases its HRas GAP activity by 25% and its Rap1 GAP activity by 76%. Conversely, phosphorylation by CDK5 increases r-synGAP's HRas GAP activity by 98% and its Rap1 GAP activity by 20%. Thus, phosphorylation by both kinases increases synGAP activity; CaMKII shifts the relative GAP activity toward inactivation of Rap1, and CDK5 shifts the relative activity toward inactivation of HRas. GAP activity toward Rap2 is not altered by phosphorylation by either kinase. CDK5 phosphorylates synGAP primarily at two sites, Ser-773 and Ser-802. Phosphorylation at Ser-773 inhibits r-synGAP activity, and phosphorylation at Ser-802 increases it. However, the net effect of concurrent phosphorylation of both sites, Ser-773 and Ser-802, is an increase in GAP activity. synGAP is phosphorylated at Ser-773 and Ser-802 in the PSD fraction, and its phosphorylation by CDK5 and CaMKII is differentially regulated by activation of NMDA-type glutamate receptors in cultured neurons.
authors.affiliation: Division of Biology and Biological Engineering.
authors.forename: Ward G
authors.initials: WG
authors.lastname: Walkup
authors.affiliation: Division of Biology and Biological Engineering.
authors.forename: Lorraine
authors.initials: L
authors.lastname: Washburn
authors.affiliation: Proteome Exploration Laboratory of the Beckman Institute, California Institute of Technology, Pasadena, California 91125.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sweredoski
authors.affiliation: Division of Biology and Biological Engineering.
authors.forename: Holly J
authors.initials: HJ
authors.lastname: Carlisle
authors.affiliation: Proteome Exploration Laboratory of the Beckman Institute, California Institute of Technology, Pasadena, California 91125.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Graham
authors.affiliation: Proteome Exploration Laboratory of the Beckman Institute, California Institute of Technology, Pasadena, California 91125.
authors.forename: Sonja
authors.initials: S
authors.lastname: Hess
authors.affiliation: Division of Biology and Biological Engineering. Electronic address: kennedym@its.caltech.edu.
authors.forename: Mary B
authors.initials: MB
authors.lastname: Kennedy
mesh_terms: D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D051360:Cyclin-Dependent Kinase 5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009474:Neurons / Q000166:cytology / Q000201:enzymology; D015513:Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013569:Synapses / Q000201:enzymology*; D020668:rap GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020703:ras GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D020690:GTPase-Activating Proteins; C528429:Hras protein, rat; D015513:Oncogene Proteins; D016194:Receptors, N-Methyl-D-Aspartate; C484739:SYNGAP1 protein, human; C484512:Syngap1 protein, mouse; C112202:Syngap1 protein, rat; D020703:ras GTPase-Activating Proteins; D051360:Cyclin-Dependent Kinase 5; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; C495905:CDK5 protein, human; C495907:Cdk5 protein, rat; C116756:RAP2A protein, human; C501009:HRAS protein, human; C495971:Hras protein, mouse; D016283:Proto-Oncogene Proteins p21(ras); D020668:rap GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins; D018631:ras Proteins
keywords: Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII); Cyclin-dependent Kinase 5 (CDK5); Mass Spectrometry (MS); Postsynaptic Density; Protein Kinase; Ras Protein; Ras-related Protein 1 (Rap1); Small GTPase; Synaptic GTPase-activating Protein (synGAP); Synaptic Plasticity