Okadaic acid-induced inhibition of B-50 dephosphorylation by presynaptic membrane-associated protein phosphatases.
The neuronal tissue-specific protein kinase C (PKC) substrate B-50 can be dephosphorylated by endogenous protein phosphatases (PPs) in synaptic plasma membranes (SPMs). The present study characterizes membrane-associated B-50 phosphatase activity by using okadaic acid (OA) and purified 32P-labeled substrates. At a low concentration of [gamma-32P]ATP, PKC-mediated [32P]phosphate incorporation into B-50 in SPMs reached a maximal value at 30 s, followed by dephosphorylation. OA, added 30 s after the initiation of phosphorylation, partially prevented the dephosphorylation of B-50 at 2 nM, a dose that inhibits PP-2A. At the higher concentration of 1 microM, a dose of OA that inhibits PP-1 as well as PP-2A, a nearly complete blockade of B-50 dephosphorylation was seen. Heat-stable PP inhibitor-2 (I-2) also inhibited dephosphorylation of B-50. The effects of OA and I-2 on B-50 phosphatase activity were additive. Endogenous PP-1- and PP-2A-like activities in SPMs were also demonstrated by their capabilities of dephosphorylating [32P]phosphorylase a and [32P]casein. With these exogenous substrates, sensitivities of the membrane-bound phosphatases to OA and I-2 were found to be similar to those of purified forms of these enzymes. These results indicate that PP-1- and PP-2A-like enzymes are the major B-50 phosphatases in SPMs.
Han
Y F
YF
Department of Biochemistry, Medical College of Ohio, Toledo 43699.
Dokas
L A
LA
D000818:Animals; D004988:Ethers, Cyclic / Q000494:pharmacology*; D019922:GAP-43 Protein; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D011919:Rats, Inbred Strains; D013570:Synaptic Membranes / Q000201:enzymology*
D004988:Ethers, Cyclic; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases