Phosphorylation of
insulin receptor substrate (IRS)
proteins on
serine residues is an important posttranslational modification that is linked to
insulin resistance. Several
phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key
receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a
serine phosphorylation site in the N-terminal
pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify
serine 24 (Ser24) as a putative substrate site for the
protein kinase C (PKC) family of
serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after
phorbol 12-myristate 13-acetate treatment of cells, and
isoform-selective inhibitor studies suggest the involvement of PKCalpha. By comparing the pharmacological characteristics of
phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKCalpha is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating
phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on
insulin-stimulated
glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal
insulin signaling and is regulated by
serine phosphorylation in a manner that could contribute to
insulin resistance.