p70 S6 Kinase Is Regulated by Protein Kinase Cζ and Participates in a Phosphoinositide 3-Kinase-Regulated Signalling Complex

Abstract

p70 S6 kinase (p70S6K) is an important regulator of cell proliferation. Its activation by growth factor requires phosphorylation by various inputs on multiple sites. Data accumulated thus far support a model whereby p70S6K activation requires sequential phosphorylations at proline-directed residues in the putative autoinhibitory pseudosubstrate domain, as well as threonine 389. Threonine 229, a site in the catalytic loop is phosphorylated by phosphoinositide-dependent kinase 1 (PDK-1). Experimental evidence suggests that p70S6K activation requires a phosphoinositide 3-kinase (PI3-K)-dependent signal(s). However, the intermediates between PI3-K and p70S6K remain unclear. Here, we have identified PI3-K-regulated atypical protein kinase C (PKC) isoform PKCζ as an upstream regulator of p70S6K. In coexpression experiments, we found that a kinase-inactive PKCζ mutant antagonized activation of p70S6K by epidermal growth factor, PDK-1, and activated Cdc42 and PI3-K. While overexpression of a constitutively active PKCζ mutant (myristoylated PKCζ [myr-PKCζ]) only modestly activated p70S6K, this mutant cooperated with PDK-1 activation of p70S6K. PDK-1-induced activation of a C-terminal truncation mutant of p70S6K was also enhanced by myr-PKCζ. Moreover, we have found that p70S6K can associate with both PDK-1 and PKCζ in vivo in a growth factor-independent manner, while PDK-1 and PKCζ can also associate with each other, suggesting the existence of a multimeric PI3-K signalling complex. This work provides evidence for a link between a phorbol ester-insensitive PKC isoform and p70S6K. The existence of a PI3-K-dependent signalling complex may enable efficient activation of p70S6K in cells.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grants GM51405 (J.B.) and CA75134 (A.T.). A.R. is a recipient of a postdoctoral fellowship award from the Juvenile Diabetes Foundation International.

We thank Shigeo Ohno for providing the PKCζ K/W construct. We also thank members of the Blenis lab for helpful discussions and for critical reading of the manuscript.

ADDENDUM IN PROOF

Following submission of this paper, an article showing that atypical protein kinase Cλ binds and regulates p70 S6 kinase was published (Akimoto et al., Biochem. J. 338:417–424, 1998).