Thyrotropin-releasing hormone stimulates MAP kinase activity in GH3 cells by divergent pathways. Evidence of a role for early tyrosine phosphorylation.
Regulation of the mitogen-activated protein (MAP) kinase by thyrotropin-releasing hormone (TRH) in GH3 rat pituitary tumor cells was investigated. Both TRH and epidermal growth factor (EGF) acutely activated this enzyme, via tyrosine and serine/threonine phosphorylation. Down-regulation of cellular protein kinase C (PKC) only partly inhibited the phosphorylation of MAP kinase by TRH, suggesting both PKC-dependent and -independent pathways. Both TRH and EGF similarly increased the phosphorylation of raf-1, by a PKC-independent mechanism. Both TRH and EGF stimulated the formation of a ras-GTP complex. This activation of ras by growth factors is thought to involve the tyrosine phosphorylation of Shc. EGF stimulated the tyrosine phosphorylation of three Shc proteins and their subsequent association with its receptor. TRH stimulated the tyrosine phosphorylation of the 52-kDa Shc protein, although neither phorbol esters nor the calcium ionophore A23187 had any effect, indicating that this effect of TRH was not dependent on PKC. Both TRH and EGF induced the association of tyrosine phosphorylated Shc proteins with a fusion protein containing SH2 and SH3 domains of Grb2, another important component in ras activation. These results provide evidence that MAP kinase is acutely activated by TRH through a PKC-dependent pathway as well as a second pathway possibly involving tyrosine phosphorylation.
Ohmichi
M
M
Department of Physiology, University of Michigan School of Medicine, Ann Arbor 48109.
Sawada
T
T
Kanda
Y
Y
Koike
K
K
Hirota
K
K
Miyake
A
A
Saltiel
A R
AR
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000494:pharmacology; D011905:Genes, ras / Q000187:drug effects; D007700:Kinetics; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D010911:Pituitary Neoplasms; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D011519:Proto-Oncogenes / Q000187:drug effects; D051381:Rats; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D019000:Phosphotyrosine; D014443:Tyrosine; D013973:Thyrotropin-Releasing Hormone; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016283:Proto-Oncogene Proteins p21(ras); D013755:Tetradecanoylphorbol Acetate