Phosphorylation of the adaptor protein SH2B1βregulates its ability to enhance growth hormone (GH)-dependent macrophage motility

Abstract

SummaryPrevious studies have shown that growth hormone (GH) recruits the adapter protein SH2B1b to the GH-activated, GH receptorassociated tyrosine kinase JAK2, implicating SH2B1b in GH-dependent actin cytoskeleton remodeling, and suggesting that phosphorylation at serines 161 and 165 in SH2B1b releases SH2B1b from the plasma membrane. Here, we examined the role of SH2B1b in GH regulation of macrophage migration. We show that GH stimulates migration of cultured RAW264.7 macrophages, and primary cultures of peritoneal and bone marrow-derived macrophages. SH2B1b overexpression enhances, whereas SH2B1 knockdown inhibits, GH-dependent motility of RAW macrophages. At least two independent mechanisms regulate the SH2B1b-mediated changes in motility. In response to GH, tyrosines 439 and 494 in SH2B1b are phosphorylated. Mutating these tyrosines in SH2B1b decreases both basal and GH-stimulated macrophage migration. In addition, mutating the polybasic nuclear localization sequence (NLS) in SH2B1b or creating the phosphomimetics SH2B1b(S161E) or SH2B1b(S165E), all of which release SH2B1b from the plasma membrane, enhances macrophage motility. Conversely, SH2B1b(S161/165A) exhibits increased localization at the plasma membrane and decreased macrophage migration. Mutating the NLS or the nearby serine residues does not alter GH-dependent phosphorylation on tyrosines 439 and 494 in SH2B1b. Mutating tyrosines 439 and 494 does not affect localization of SH2B1b at the plasma membrane or movement of SH2B1b into focal adhesions. Taken together, these results suggest that SH2B1b enhances GH-stimulated macrophage motility via mechanisms involving phosphorylation of SH2B1b on tyrosines 439 and 494 and movement of SH2B1b out of the plasma membrane (e.g. as a result of phosphorylation of serines 161 and 165).

The Effect of SH2B1 Variants on Expression of Leptin- and Insulin-Induced Pathways in Murine Hypothalamus

“…Although nuclearization of SH2B1 has been shown for the beta, but not the alpha, splice variant of the gene, the differential expression pattern does not have to be a direct outcome of SH2B1 acting as a transcription factor. Whether SH2B1 has to bind specific partners for nuclear shuttling is still unclear [26], although binding of STATs could well be one of them [42]. …”

SH2B1 regulation of energy balance, body weight, and glucose metabolism

“…Both the N-terminal region (amino acids and the SH2 domain of SH2B1β are required for GH stimulation of cell motility [68] . Additionally, SH2B1β mutants lacking Tyr 439 and Tyr 494 phosphorylation sites are unable to enhance GH-stimulated membrane ruffling in 3T3-F442A fibroblasts [23] and GH-stimulated motility of RAW264.7 macrophages [56] . SH2B1-Rac interaction is involved in mediating GH-promoted actin cytoskeletal reorganization and cell motility [68] .…”

“…GH increases the cycling of SH2B1 into and out of focal adhesions [26] , and promotes SH2B1 colocalization with F-actin in membrane ruffles [66] . Overexpression of SH2B1β, but not SH2 domain-defective SH2B1β(R555E), enhances the ability of GH to stimulate both membrane ruffles in 3T3-F442A fibroblasts [59,66] and macrophage migration [56] . In fact, SH2B1β(R555E) blocks GH-induced lamellipodia dynamics in 3T3-F442A cells [68] .…”

“…GH stimulates the binding of SH2B1 to JAK2 and robust tyrosine phosphorylation of SH2B1 by JAK2 in 3T3-F442A fibroblasts [12] . GH stimulates JAK2-mediated phosphorylation of SH2B1 on Tyr 439 and Tyr 494 residues [56] . Like leptin, GH stimulates phosphorylation of JAK2 on Tyr 813 which binds to the SH2 domain of SH2B1 [57] .…”

Minireview: Mechanisms of Growth Hormone-Mediated Gene Regulation

“…Finally, an additional adaptor protein that is phosphorylated by JAK2 with GH is SH2B1␤ (74). The activated protein functions in actin cytoskeleton remodeling that underlies GH-dependent macrophage mobility (75), illustrating another of the diverse effects of GH.…”