Mitogenic signaling of thrombin in mesangial cells: role of tyrosine phosphorylation.
Thrombin elicits multiple biological effects on a variety of cells. We have previously shown that thrombin is a potent mitogen for human glomerular mesangial cells. This mitogenic effect of thrombin is associated with activation of phospholipase C (PLC) and induction of platelet-derived growth factor (PDGF) gene expression. The thrombin receptor, which belongs to the guanine nucleotide binding protein (G protein)-coupled receptor family, has recently been shown to induce rapid tyrosine phosphorylation of cellular proteins. In the present study, we investigated the role of protein-tyrosine phosphorylation in mediating the cellular responses elicited by thrombin in human glomerular mesangial cells. Amino acid labeling followed by immunoprecipitation with phosphotyrosine antibodies demonstrate that thrombin stimulates tyrosine phosphorylation of a set of cellular proteins. Treatment of mesangial cells with thrombin followed by immunoblotting with phosphotyrosine antibodies showed three major bands of tyrosine-phosphorylated proteins approximately 130, 70, and 44-42 kDa. Phosphorylation of these proteins was inhibited by two tyrosine kinase inhibitors, herbimycin A and genistein. Both compounds inhibited DNA synthesis and PDGF B-chain gene expression but had no effect on inositol phosphates production or increases in cytosolic calcium in response to thrombin. These data demonstrate that protein-tyrosine phosphorylation is not required for thrombin-induced PLC activation with inositol phosphate formation and subsequent intracellular calcium release, but it is an absolute requirement for thrombin-induced DNA synthesis and PDGF B-chain gene expression.
Grandaliano
G
G
Department of Medicine, University of Texas Health Science Center at San Antonio.
Choudhury
G G
GG
Biswas
P
P
Abboud
H E
HE
D016227:Benzoquinones; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D015870:Gene Expression / Q000187:drug effects; D019833:Genistein; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007295:Inositol Phosphates / Q000378:metabolism; D007529:Isoflavones / Q000494:pharmacology; D047029:Lactams, Macrocyclic; D008934:Mitogens / Q000494:pharmacology*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D010982:Platelet-Derived Growth Factor / Q000096:biosynthesis* / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D017828:Rifabutin / Q000031:analogs & derivatives; D013462:Sulfur Radioisotopes; D013917:Thrombin / Q000494:pharmacology*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism
D016227:Benzoquinones; D007295:Inositol Phosphates; D007529:Isoflavones; D047029:Lactams, Macrocyclic; D008934:Mitogens; D010750:Phosphoproteins; D010982:Platelet-Derived Growth Factor; D011809:Quinones; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D017828:Rifabutin; D019000:Phosphotyrosine; D014443:Tyrosine; C020754:herbimycin; D004247:DNA; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D013917:Thrombin; D003545:Cysteine; D002118:Calcium