Fig. 3.

Functional link between ROCK isoforms and IRS-1. A, Physical interaction between ROCK isoforms and IRS-1 in culture cell lines and mouse muscle. Cell lysates from 3T3-L1 adipocytes, L6 myoblasts, mouse muscle tissue, and HEK-293T were subjected to immunoprecipitation (IP) with a ROCK1 (R1), ROCK2 (R2), or IRS-1 antibody. Immunoprecipitated proteins were resolved by PAGE, and ROCK and IRS-1 were visualized by immunoblotting with a ROCK1, ROCK2, or IRS-1 antibody. C, Effects of IRS-1 serine 632/635 phosphorylation on insulin-stimulated glucose transport and PI3K activation in 3T3-L1 adipocytes. Cells were transduced with recombinant adenovirus encoding β-Gal, WT-IRS-1, S632/635E-IRS-1 (S/E), or S632/635A-IRS-1 (S/A) as described in Materials and Methods. IRS-1 was visualized by immunoblotting with a polyclonal IRS-1 antibody. Cells were serum starved and then stimulated with insulin, as indicated, for 30 min. [³H]-2-deoxy-D-glucose uptake (B) and PI3K activity (C) was measured, and the results are expressed as a percentage of basal glucose transport in control cells. Data are means ± sem; #, P < 0.01 vs. the corresponding condition with WT-IRS-1; *, P < 0.01 vs. the corresponding condition with β-Gal.