Phosphatidic acid activation of protein kinase C-ζ overexpressed in COS cells. Comparison with other protein kinase C isotypes and other acidic lipids

C. Limatola, D. Schaap, W. H. Moolenaar, W. J. Van Blitterswijk

Research output: Contribution to journalArticlepeer-review

Abstract

Phosphatidic acid (PA) is produced rapidly in agonist-stimulated cells, but the physiological function of this PA is unknown. We have examined the effects of PA on distinct isoforms of protein kinase C (PKC) using a new cell-free assay system. Addition of PA to cytosol from COS cells overexpressing PKC-α, ε or -ζ differentially activated all three isotypes, as shown by PKC autophosphorylation, and prominent phosphorylation of multiple endogenous substrates. In the absence of Ca2+, the diacylglycerol-insensitive ζ-isotype of PKC was most strongly activated by both PA and bisPA, a newly identified product of activated phospholipase D, with each lipid inducing its own profile of protein phosphorylation. BisPA was also a strong activator of PKC-ε, but a weak activator of PKC-α. Ca2+, at ≥ 0.1 μM, inhibited PA and bisPA activation of PKC-ζ, but did not affect PKC-ε activation. In contrast, PKC-α was strongly activated by PA only in the presence of Ca2+. BisPA-induced phosphorylations mediated by PKC-ζ could be mimicked in part by other acidic phospholipids and unsaturated fatty acids. PA activation of PKC-ζ was unique in that PA not only stimulated PKC-ζ-mediated phosphorylation of distinctive substrates, but also caused an upward shift in electrophoretic mobility of PKC-ζ, which was not observed with other acidic lipids or with PKC-α or -ε. We have presented evidence that this mobility shift is not caused by PKC-ζ autophosphorylation, but it coincides with physical binding of PA to PKC-ζ. These results suggest that in cells stimulated under conditions where intracellular Ca2+ is at (or has returned to) basal level, PA may be a physiological activator of PKC-ζ.

Original languageEnglish
Pages (from-to)1001-1008
Number of pages8
JournalBiochemical Journal
Volume304
Issue number3
Publication statusPublished - 1994

ASJC Scopus subject areas

  • Biochemistry

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