SH2-B is required for growth hormone-induced actin reorganization

The Src homology-2 (SH2) domain-containing protein SH2-B beta is a substrate of the growth hormone (GH) receptor-associated tyrosine kinase JAK2. Here we tested whether SH2-B beta is involved in GH regulation of the actin cytoskeleton. Based on cell fractionation and confocal microscopy, we find SH2-B beta present at the plasma membrane and in the cytosol. SH2-B beta colocalized with filamentous actin in GH and platelet-derived growth factor (PDGF)-induced membrane ruffles. To test if SH2-B beta is required for actin reorganization, we transiently overexpressed wild-type or mutant SH2-B beta in 3T3-F442A cells and assayed for GH- and PDGF-induced membrane ruffling and fluid phase pinocytosis. Overexpression of wild-type SH2-B beta enhanced ruffling and pinocytosis produced by submaximal GH but not sub-maximal PDGF. Point mutant SH2-B beta (R555E) and truncation mutant Delta C555, both lacking a functional SH2 domain, inhibited membrane ruffling and pinocytosis induced by GH and PDGF. Mutant Delta N504, which possesses a functional SH2 domain and enhances JAK2 kinase activity in overexpression systems, also inhibited GH stimulated membrane ruffling. Delta N504 failed to inhibit GH-induced nuclear localization of Stat5B, indicating JAK2 is active in these cells. Taken together, these results show that SH2-B beta is required for GH-induced actin reorganization by a mechanism discrete from the action of SH2-B beta as a stimulator of JAK2 kinase activity.