Differential activation of adenylyl cyclase by protein kinase C isoenzymes

J Kawabe,G Iwami,T Ebina,S Ohno,T Katada,Y Ueda,C.J Homcy,Y Ishikawa

doi:10.1016/s0021-9258(19)89424-3

J Kawabe, G Iwami + Show 6 more

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https://doi.org/10.1016/s0021-9258(19)89424-3

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Journal: Journal of Biological Chemistry	Publication Date: Jun 1, 1994
Citations: 242	License type: cc-by

Affiliation: Columbia University

Abstract

Cyclic AMP production within cells is altered upon protein kinase C (PKC) activation; however, whether PKC directly modulates adenylyl cyclase (AC) catalytic activity has been controversial. Molecular studies have elucidated the existence of multiple PKC isoenzymes although the functional role of this diversity is not clear. Using purified PKC and AC isoenzymes, we demonstrate that PKC zeta directly phosphorylates type VAC, leading to an approximate 20-fold increase in its catalytic activity, a significantly larger enhancement than that achieved with forskolin (approximately 5-fold), the most potent activator of AC. When forskolin and PKC phosphorylation are combined, type V AC catalytic activity is increased 100-fold over basal levels. The two PKC isoenzymes (alpha and zeta) are additive in their capacity to activate AC, although PKC alpha is less potent than PKC zeta. Our data indicate that PKC can directly and potently regulate AC activity in an isoenzyme-specific manner, suggesting that direct cross-talk plays a major role in coordinating the activity of these two principal signal transduction pathways.

Highlights

Cyclic AMP production within cells is altered upon the CAMPpathway
Molecularstudies have binant type V adenylyl cyclase (AC) and protein kinase C (PKC) isoenzymes, we demonstrate that elucidated the existence of multiple PKC isoenzymes al- PKC modifies the catalytic activity of type V AC directly and though the functional roleof this diversity is not clear. potently in an isoenzyme-specific manner
Using purifiedPKC andAC isoenzymes, we demonstrate that PKC5 directly phosphorylates typVeAC, leading to an approximate 20-foldincrease in its catalytic activity, a significantly larger enhancement than that achieved with forskolin (“dfold), the most potent activatorAoCf

Summary

Differential Activationof Adenylyl Cyclase by Protein KinaseC Isoenzymes*

(Received for publication, January 6, 1994, and inrevised form, February 25, 1994). Jun-ichi KawabeS, Gensho Iwamis, Toshiaki EbinaS, Shigeo Ohnon, Toshiaki Katadall, Yoshihiko Uedan, Charles J. The agarose wasboiled in catalytic activity of AC were enhanced still further over that the presence of Laemmli buffer and analyzed by SDS-PAGE After separating on 8% SDS-PAGE, the phosphorylatedtype V AC was excised and was similar to that produced by PKCS alone (Fig. 3) Such cumulative enhancement inAC catalytic activity suggests that the two isoenzymes modify the AC molecule differently. Type V AC was firstmaximally phosphorylated with each isoenzyme, and was completely digestedwith trypsin.The proteolytic products were analyzed using acidic PAGE in order to separate small peptide fragments [17].

AND DISCUSSION

Activation of Adenylyl Cyclase by Protein Kinase C Isoenzymes a

Activation of AdenyClyylclase by ProteKininIasCsoeenzymes