/oioii~l ü/ /VelfIQ(/,C/iIiSl/V I Appirlcott—Ra\ en I’ hlkher6, Philadelphia I ‘)‘)6 I Ilternil a cal Society for Neurochemi 6try Modulation of ~ -Protein Kinase C by Cyclic AMP in PC 12 Cells Occurs Through Phosphorylation by Protein Kinase A Marie W. Wooten, M. Lamar Seibenhener, Laura H. Matthews, Guisheng Zhou, and *Elajne S. Coleman Department oj’ Zoology, Auburn University, Auburn; and * Department qt Anatomy, School of Veterinary Medicine, Tuskegee University, Tuskegee, Alabama, U.S.A. Abstract: Although cyclic AMP (cAMP) has been re- ported to cross talk with the protein kinase C (PKC) sys- tem, effects of elevated intracellular cAMP on the activi- ties of specific PKC isoforms have not been studied. We report findings from a permeabilized cell assay that was used to examine changes in the activity of the atypical PKC isoforms brought about by exposure of PC12 cells to agents that elevate intracellular cAMP. We found that increases in intracellular cAMP led to rapid stimulation of atypical PKC activity, 40—70% above control, for a sustained period of time, a response that occurred inde- pendent of the phorbol 12-myristate 13-acetate (PMA)- sensitive PKC isoforms. Changes in intracellular cAMP levels resulted in a dose-dependent redistribution of ~- PKC to the cytoplasm with a concomitant increase in the phosphorylation state of the enzyme. Incubation of purified 1-PKC with increasing concentrations of PKA likewise caused a twofold increase in the phosphorylation state of ~-PKC. In contrast to the positive effect that PKA-mediated phosphorylation had on the activity of ~- PKC, the enzyme displayed reduced binding to ras when phosphorylated. Taken together, these findings are con- sistent with the hypothesis that protein phosphorylation of PKC acts as a positive effector of its enzyme activity and may serve as a negative modulator for interaction with other proteins. Key Words: PC12 cells—Protein ki- nase C isoforms—Cyclic AMP—Cross talk—ras. J. Neurochem. 67, 1023—1031 (1996). multiple PKC isoforms, i.e., a, ~, y, 6, , and ç (Wooten et a!., 1992). It has been documented that NGF treatment of PC 12 cells results in translocation as well as activation of PKC isoforms (Ohmichi et al., 1993; Wooten et al., 1994). More important, in cells deficient of the phorbol 12-myristate 13-acetate (PMA)-sensitive PKC isoforms (a, ~, y, 6, and ), NGF translocates the PMA-insensitive isoform-ç (Wooten et al., 1994). Removal of ~-PKC from cells deficient in PMA-sensitive isoforms inhibits NOF-in- duced neurite outgrowth (Coleman and Wooten, 1994). Despite these observations, the precise role for individual PKC isoforms within differentiation signal- ing cascades of PCI2 cells remains poorly studied. In other systems, it has been shown that ç-PKC operates in a cooperative fashion with ras and has been postulated to integrate signals from both phosphatidyl- choline—phospholipase C and phosphatidylinositol-3- kinase pathways (Berra et al., 1993; Nakanishi et al., 1993), acting upstream of mitogen-activated protein (MAP) kinase. In addition, ~-PKC has been reported to bind directly to ras (Diaz-Meco et al., 1994b). In fibroblasts, elevated cyclic AMP (cAMP) levels mod- ulate ras function, resulting in inhibition of MAP ki- nase (Wu et al., 1993), whereas elevated cAMP levels The pheochromocytoma cell line (PCI2) has been used extensively to unravel steps leading to neuronal differentiation. These cells express receptors for and respond to nerve growth factor (NGF). In the presence ui NGF. PC 12 cells differentiate into sympathetic neu- runs. Protein kinase C (PKC) is one protein phosphor- ylation system activated in response to NGF (Hama ci al., 1986; Heasley and Johnson, 1989; Kondratyev vial., 1990; Ohmichi et al., 1993; Wooten et al., 1994). PKC is composed of a family of highly homokgous isolorm proteins, the products of discrete genes whose members are categorized as classic, nonelassic, and atypical (Hug and Sarre, 1993). PCI2 cells express Received February 2, 1996; revised manuscript received March 17, 996: accepted April 22, 1996. Address correspondence and reprint requests to Dr. M. W. Wuoten at 331 Funchess Hall, Department of Zoology, Auburn University, Auburn, AL 36849-5414, U.S.A. Abbreviations used: aPKC, phorbol ester insensitive/atypical pro- tein kinase C; 8-Br-cAMP, 8-Br-cyclic AMP; cAMP, cyclic AMP; 8-Cl-cAMP, 8-(4-chlurophenylihio)-cAMP; cPKC, Ca 2 -sensitive/ phospholipid-dependent protein kinase C; CT, cholera toxin; dBcAMP, N6, 2 ‘-O-dibutyryl-cAMP; dBcGMP, N6. 2 ‘-O-dibutyryl- cGMP; TBMX, isubutylmetliylxanthine; MAP kinase, mitogen-acti- vated protein kinase; MEK, MAP kinase kinase; NGF, nerve growth factor; PACAP-38, pituitary adenylate cyclase—activating polypep- tide 38; PAGE, polyacrylamide gel electruphoresis; PBS. phosphate-S buffered saline; PKA, cAMP-dependent protein kinase: PKC, pro- tein kinase C; PKI, protein kinase I; PMA, phorhol 12-myristate 13- acetate; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate. 1023