SH2-B is required for growth hormone-induced actin reorganization.
The Src homology-2 (SH2) domain-containing protein SH2-Bbeta is a substrate of the growth hormone (GH) receptor-associated tyrosine kinase JAK2. Here we tested whether SH2-Bbeta is involved in GH regulation of the actin cytoskeleton. Based on cell fractionation and confocal microscopy, we find SH2-Bbeta present at the plasma membrane and in the cytosol. SH2-Bbeta colocalized with filamentous actin in GH and platelet-derived growth factor (PDGF)-induced membrane ruffles. To test if SH2-Bbeta is required for actin reorganization, we transiently overexpressed wild-type or mutant SH2-Bbeta in 3T3-F442A cells and assayed for GH- and PDGF-induced membrane ruffling and fluid phase pinocytosis. Overexpression of wild-type SH2-Bbeta enhanced ruffling and pinocytosis produced by submaximal GH but not submaximal PDGF. Point mutant SH2-Bbeta (R555E) and truncation mutant DeltaC555, both lacking a functional SH2 domain, inhibited membrane ruffling and pinocytosis induced by GH and PDGF. Mutant DeltaN504, which possesses a functional SH2 domain and enhances JAK2 kinase activity in overexpression systems, also inhibited GH-stimulated membrane ruffling. DeltaN504 failed to inhibit GH-induced nuclear localization of Stat5B, indicating JAK2 is active in these cells. Taken together, these results show that SH2-Bbeta is required for GH-induced actin reorganization by a mechanism discrete from the action of SH2-Bbeta as a stimulator of JAK2 kinase activity.
Herrington
J
J
Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622, USA.
Diakonova
M
M
Rui
L
L
Gunter
D R
DR
Carter-Su
C
C
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D013006:Growth Hormone / Q000378:metabolism; D007150:Immunohistochemistry; D053614:Janus Kinase 2; D051379:Mice; D018613:Microscopy, Confocal; D008894:Milk Proteins; D010873:Pinocytosis; D010957:Plasmids; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism; D014162:Transfection
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D008894:Milk Proteins; D010982:Platelet-Derived Growth Factor; D011518:Proto-Oncogene Proteins; D050799:STAT5 Transcription Factor; C115327:Sh2bpsm1 protein, mouse; C494096:Stat5b protein, mouse; D015534:Trans-Activators; D013006:Growth Hormone; D011505:Protein-Tyrosine Kinases; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2