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.
Highlights
SynGAP inactivates Ras and Rap at synapses
Purification of r-synGAP—It was previously shown that a shortened form of synGAP comprising residues 103–725 can be expressed in soluble form in E. coli and that this construct displays GTPase-activating protein (GAP) activity against Rap with a potency similar to other Rap-GAPs
A combination of immobilized metal affinity chromatography and SEC was used to purify the synGAP constructs as described under “Experimental Procedures.” sr-synGAP was purified to Ͼ95% purity, with typical yields in excess of 75%. r-synGAP was purified by immobilized metal affinity chromatography alone to ϳ70% purity, with yields in excess of 85%. r-synGAP eluted in the void volume of SEC columns with no improvement in purity, suggesting that the presence of the disordered domain and/or the coiled-coil domain may predispose r-synGAP to multimerization
Summary
Expression, and Purification of r-synGAP—Soluble recombinant synGAP comprising residues 103–1293 (r-synGAP) was purified from Escherichia coli. Phosphorylated or nonphosphorylated synGAP (250 – 500 nM) was mixed with increasing concentrations of each GTPase bound to radioactive [␥-32P]GTP at 25 °C in 6.5 l of Assay Buffer (25 mM Tris, pH 8.0, 100 mM NaCl, 15 mM MgCl2, and 5 mM DTT) as described previously (15). The solubilized fraction was brought to 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1ϫ Roche Applied Science protease inhibitor tablet, 0.2% deoxycholate, and the protein concentration was determined by the bicinchoninic acid method To measure phosphorylation of synGAP, 15–30 g of cell lysate was fractionated by SDS-PAGE, transferred to nitrocellulose membranes, blocked, and incubated with primary antibodies as described above Antibodies included those listed above and ␣-MAP-2 Reported protein values were normalized either for staining for total synGAP or for MAP-2, as stated
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