Interruption of growth hormone signaling via SHC and ERK in 3T3-F442A preadipocytes upon knockdown of insulin receptor substrate-1.
Insulin receptor substrate-1 (IRS-1) is a docking protein tyrosine phosphorylated in response to insulin, IGF-1, GH, and other cytokines. IRS-1 has an N-terminal plekstrin homology domain (which facilitates membrane localization), a phosphotyrosine-binding domain [which associates with tyrosine-phosphorylated insulin receptor or IGF-1 receptor (IGF-1R)], and tyrosine residues that, when phosphorylated, bind signaling molecules. The role of IRS-1 in GH signaling is uncertain. We previously reported that IRS-1 and Janus kinase 2 associate independently of tyrosine phosphorylation via IRS-1's N terminus and that IRS-1 reconstitution greatly enhances GH-induced ERK, but not STAT5, activation. We now use GH-responsive 3T3-F442A preadipocytes to study the influence of IRS-1 on GH action. We stably transfected cells with vector only (Control) or a vector encoding IRS-1 short hairpin RNA [knockdown (KD)] and compared representative clones. Immunoblotting confirmed more than 80% knockdown of IRS-1 in KD cells. GH caused characteristic Janus kinase 2 and STAT5 activation in both Control and KD cells, but ERK activation was dramatically reduced in KD cells in GH time course and dose-response experiments. Notably, GH-induced Src homology collagen (SHC) activation and SHC-Grb2 association in KD cells were also markedly diminished compared with Control cells. Subcellular fractionation revealed that IRS-1 in Control cells was largely cytosolic, but the component isolated with plasma membranes was highly enriched in lipid raft membranes (LR). In KD cells, GH-induced ERK activation in the LR fraction was particularly diminished compared with Control cells. These data suggest that LR-enriched IRS-1 contributes substantially to GH-induced ERK activation in LR in 3T3-F442A fibroblasts. Furthermore, our results are consistent with IRS-1 residing upstream of SHC in the GH-induced ERK-signaling pathway.
Wang
Xiangdong
X
University of Alabama at Birmingham, Birmingham, Alabama 35294-0012, USA.
Yang
Ning
N
Deng
Luqin
L
Li
Xin
X
Jiang
Jing
J
Gan
Yujun
Y
Frank
Stuart J
SJ
D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology / Q000502:physiology; D000818:Animals; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D013006:Growth Hormone / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins / Q000235:genetics / Q000378:metabolism*; D053614:Janus Kinase 2 / Q000235:genetics / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D011986:Receptors, Somatotropin / Q000235:genetics / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000235:genetics / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D055504:Insulin Receptor Substrate Proteins; D011986:Receptors, Somatotropin; D050799:STAT5 Transcription Factor; D055765:Shc Signaling Adaptor Proteins; D013006:Growth Hormone; D053614:Janus Kinase 2; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases