In vitro phosphorylation of insulin receptor substrate 1 by protein kinase c-zeta: Functional analysis and identification of novel phosphorylation sites

作者：Sommerfeld MR; Metzger S; Stosik M; Tennagels N; Eckel J^*

来源：Biochemistry, 2004, 43(19): 5888-5901.

DOI：10.1021/bi049640v

Protein kinase C-zeta (PKC-zeta) participates both in downstream insulin signaling and in the negative feedback control of insulin action. Here we used an in vitro approach to identify PKC-zeta phosphorylation sites within insulin receptor substrate I (IRS-1) and to characterize the functional implications. A recombinant IRS-1 fragment (rIRS-1(449-664)) Containing major tyrosine motifs for interaction with phosphatidylinositol (PI) 3-kinase strongly associated to the p85alpha subunit of PI 3-kinase after Tyr phosphorylation by the insulin. receptor. Phosphorylation of rlRS-1(449-664) by PKC-zeta induced a prominent inhibition of this process with a mixture of classical PKC isoforms being less effective. Both PKC-zeta and the classical isoforms phosphorylated rIRS-1(449-664) on Ser(612). However, modification of this residue did not reduce the affinity of p85alpha binding to pTyr-containing peptides (amino acids 605-615 of rat IRS-1), as determined by surface plasmon resonance. rIRS-1(449-664) was then phosphorylated by PKC-zeta using [P-32]ATP and subjected to tryptic phosphopeptide mapping based on two-dimensional HPLC coupled to mass spectrometry. Ser(498) and Ser(570) were identified as novel phosphoserine sites targeted by PKC-zeta. Both sites were additionally confirmed by phosphopeptide mapping of the corresponding Ser --> Ala mutants of rlRS-1(449-664). Ser570 was specifically targeted by PKC-zeta, as shown by immunoblotting with a phosphospecific antiserum against Ser(570) of IRS-1. Binding of p85alpha to the S570A mutant was less susceptible to inhibition by PKC-zeta, when compared to the S612A mutant. In conclusion, our in vitro data demonstrate a strong inhibitory action of PKC-zeta at the level of IRS-1/PI 3-kinase interaction involving multiple serine phosphorylation sites. Whereas Ser(612) appears not to participate in the negative control of insulin signaling, Ser(570) may at least partly contribute to this process.

• 出版日期2004-5-18