Figure 4.
Overexpression of PKC-ζ inhibits insulin-stimulated tyrosine phosphorylation of IRS-3. NIH-3T3IR cells were transiently cotransfected with HA-tagged IRS-1 or myc-tagged IRS-3 and either an empty control vector or PKC-ζ. Transfected cells were treated with 100 nm insulin for 0, 2, or 60 min. A, Experiments were as described in legend to Fig. 3A. B, Cell lysates were immunoprecipitated (IP) with anti-myc antibody and subsequently immunoblotted (IB) with antiphosphotyrosine (top panel) or anti-IRS-3 antibody (bottom panel). Results from five independent experiments were quantified by scanning densitometry and expressed as mean ± sem of tyrosine phosphorylated IRS protein normalized to total IRS protein. Overexpression of PKC-ζ significantly reduced insulin-stimulated tyrosine phosphorylation of both IRS-1 and IRS-3 at 2 and 60 min when compared with control cells (*, P = 0.03; §, P = 0.03; ¶, P = 0.03; #, P = 0.04).