12-457 Sigma-AldrichMYPT1 (654-880)

Cellular responses are determined by a number of signaling cues in the local microenvironment, such as growth factors and extracellular matrix (ECM). In cultures of mammary epithelial cells (MECs), functional differentiation requires at least two types of signal, lactogenic hormones (i.e., prolactin, insulin, and hydrocortisone) and the specialized ECM, basement membrane (BM). Our previous work has shown that ECM affects insulin signaling in mammary cells. Cell adhesion to BM promotes insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and association of PI3K with IRS-1, whereas cells cultured on stromal ECM are inefficient in transducing these post-receptor events. Here we examine the mechanisms underlying ECM control of IRS phosphorylation. Compared to cells cultured on BM, cells on plastic exhibit higher level of RhoA activity. The amount and the activity of Rho kinase (Rok) associated with IRS-1 are greater in these cells, leading to serine phosphorylation of IRS-1. Expression of dominant negative RhoA and the application of Rok inhibitor Y27632 in cells cultured on plastic augment tyrosine phosphorylation of IRS-1. Conversely, expression of constitutively active RhoA in cells cultured on BM impedes insulin signaling. These data indicate that RhoA/Rok is involved in substratum-mediated regulation of insulin signaling in MECs, and under the conditions where proper adhesion to BM is missing, such as after wounding and during mammary gland involution, insulin-mediated cellular differentiation and survival would be defective.

It is clear from several studies that myosin phosphatase (MP) can be inhibited via a pathway that involves RhoA. However, the mechanism of inhibition is not established. These studies were carried out to test the hypothesis that Rho-kinase (Rho-associated kinase) via phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) inhibited MP activity and to identify relevant sites of phosphorylation. Phosphorylation by Rho-kinase inhibited MP activity and this reflected a decrease in V(max). Activity of MP with different substrates also was inhibited by phosphorylation. Two major sites of phosphorylation on MYPT1 were Thr(695) and Thr(850). Various point mutations were designed for these phosphorylation sites. Following thiophosphorylation by Rho-kinase and assays of phosphatase activity it was determined that Thr(695) was responsible for inhibition. A site- and phosphorylation-specific antibody was developed for the sequence flanking Thr(695) and this recognized only phosphorylated Thr(695) in both native and recombinant MYPT1. Using this antibody it was shown that stimulation of serum-starved Swiss 3T3 cells by lysophosphatidic acid, thought to activate RhoA pathways, induced an increase in Thr(695) phosphorylation on MYPT1 and this effect was blocked by a Rho-kinase inhibitor, Y-27632. In summary, these results offer strong support for a physiological role of Rho-kinase in regulation of MP activity.