Identification and Characterization of Protein Kinase FA/ Glycogen Synthase Kinase 3 in Clathrin-Coated Brain Vesicles

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Abstract
[[abstract]]Mg-ATP-dependent protein phosphatase activating factor [kinase F(A)/glycogen synthase kinase 3 (GSK-3)] has been identified in highly purified clathrin-coated vesicles (CCVs) isolated from pig brain. Kinase F(A) was found to exist in an inactive state but can be activated by 1% Triton X-100 or 1 M Tris-HCI extraction in brain CCVs. Activation of kinase F(A) in CCVs is due to disassociation of the kinase from CCVs as demonstrated on sucrose density-gradient ultracentrifugation and Sepharose CL-4B gel filtration. Using purified brain CCVs as substrates, kinase F(A) enhanced the endogenous phosphorylation of assembly protein complexes in the molecular weight range of 100,000-130,000 severalfold, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. Comparisons with well-defined brain CCV-associated endogenous protein kinases such as pp50 kinase/AP50 and casein kinase 2 provide evidence that kinase F(A)/GSK-3 represents a third potent and unique CCV-associated protein kinase distinctly different from the previously described CCV protein kinases, suggesting the possible involvement of kinase F(A) in the regulation of CCV functions in the brain. The results also support the notion that protein kinase F(A) is involved in cell surface signal transduction in the CNS.[[fileno]]2050116010054[[department]]生科