title: A role for Grb2-associated binder-1 in growth hormone signaling.
abstract: GH signaling begins with activation of the GH receptor (GHR)-associated cytoplasmic tyrosine kinase, Janus kinase-2. GH-induced Janus kinase-2 activation leads to engagement of several signaling pathways, including the extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase, phosphoinositol 3-kinase, and signal transducer and activator of transcription-5 (STAT5) pathways. Previous work suggests that ERK activation in response to GH may be modulated by several proteins acting as docking molecules, including the epidermal growth factor receptor (EGFR) and insulin receptor substrate-1. In this study we investigate potential roles for the pleckstrin homology (PH) domain-containing insulin receptor substrate-like protein, Grb-2-associated binder-1 (Gab1), in GH signaling. We find in 3T3-F442A preadipocytes that GH promotes tyrosine phosphorylation of Gab1 and its association with SHP2, an Src homology 2-containing cytoplasmic tyrosine phosphatase. The Grb2 adapter protein, in contrast, is specifically coimmunoprecipitated with Gab1, even in the absence of GH exposure. Using a COS-7 cell transient reconstitution system, we observed that GH-induced Gab1 tyrosine phosphorylation is dependent on the Gab1 PH domain, whereas GH-induced coimmunoprecipitation of SHP2 requires tyrosine 627 of Gab1, as previously reported for EGF-induced Gab1-SHP2 association. Deletion of the Gab1 PH domain significantly attenuates GH-induced ERK activation and trans-activation of a c-fos enhancer-driven reporter construct compared with wild-type Gab1 in this system. In contrast, GH-induced STAT5 tyrosine phosphorylation and STAT5-dependent trans-activation are similar in cells expressing wild-type or PH domain-deleted Gab1. Notably, neither the ERK nor the STAT5 GH-dependent signaling outcome is affected by expression of the Gab1 mutant with tyrosine 627 changed to phenylalanine. Finally, we observed GH-dependent translocation of a wild-type, but not a PH domain-deleted, Gab1-green fluorescent protein chimera from the cytoplasm to the plasma membrane. Our results suggest selective involvement of Gab1 in GH-induced ERK activation and implicate the Gab1 PH domain as critical in this involvement.
authors.affiliation: Department of Medicine, Division of Endocrinology and Metabolism, University of Alabama, Birmingham, Alabama 35294, USA.
authors.forename: Sung-Oh
authors.initials: SO
authors.lastname: Kim
authors.forename: Kimberly
authors.initials: K
authors.lastname: Loesch
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Wang
authors.forename: Jing
authors.initials: J
authors.lastname: Jiang
authors.forename: Lin
authors.initials: L
authors.lastname: Mei
authors.forename: Jess M
authors.initials: JM
authors.lastname: Cunnick
authors.forename: Jie
authors.initials: J
authors.lastname: Wu
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Frank
mesh_terms: D016475:3T3 Cells / Q000737:chemistry; D048868:Adaptor Proteins, Signal Transducing; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D019556:COS Cells; D004789:Enzyme Activation / Q000187:drug effects; D049452:Green Fluorescent Proteins; D015801:Helminth Proteins / Q000378:metabolism; D019382:Human Growth Hormone / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D053614:Janus Kinase 2; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D016296:Mutagenesis; D010750:Phosphoproteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D011817:Rabbits; D011986:Receptors, Somatotropin / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C098587:GAB1 protein, human; C490897:Gab1 protein, mouse; D015801:Helminth Proteins; D008164:Luminescent Proteins; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D011986:Receptors, Somatotropin; D011993:Recombinant Fusion Proteins; D019382:Human Growth Hormone; D049452:Green Fluorescent Proteins; C095048:microfilarial sheath protein, Helminth; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2; D019950:Mitogen-Activated Protein Kinase 1