Autocrine transforming growth factor alpha regulates cell adhesion by multiple signaling via specific phosphorylation sites of p70S6 kinase in colon cancer cells.
Recently, we showed that autocrine transforming growth factor alpha (TGFalpha) controls the epidermal growth factor receptor (EGFR)-mediated basal expression of integrin alpha2, cell adhesion and motility in highly progressed HCT116 colon cancer cells. We also reported that the expression of basal integrin alpha2 and its biological effects are critically controlled by the constitutive activation of the ERK/MAPK pathway (Sawhney, R. S., Sharma, B., Humphrey, L. E., and Brattain, M. G. (2003) J. Biol. Chem. 278, 19861-19869). In the present report, we further examine the downstream signaling mechanisms underlying EGFR/ERK signaling and integrin alpha2 function in HCT116 cells. Selective MEK inhibitors attenuated TGFalpha-mediated basal activation of p70S6K (S6K) specifically at Thr-389, indicating that this S6K site is downstream of ERK/MAPK signaling. Cells were treated with the selective protein kinase C (PKC) inhibitor bisindolylmaleimide to determine the role of PKC in S6K activation. The Thr-421 and Ser-424 phosphorylation sites of S6K were specifically inhibited by bisindolylmaleimide, which also blocked integrin alpha2 expression, cell adhesion, and motility. These data establish a novel cell motility function of S6K via PKC activation in a cancer cell. In addition, we examined whether mammalian target of rapamycin signaling controls S6K activation. Rapamycin inhibited constitutive S6K phosphorylation specifically at Thr-389, Thr-421, and Ser-424 sites. The assignment of these phosphorylation sites on S6K to biological functions was unequivocally confirmed by transfection of cells with specific single phosphorylation site dominant negative mutants. These experiments show for the first time that autocrine TGFalpha regulates cell adhesion function by multiple signaling pathways via specific phosphorylation sites of S6K in cancer cells.
Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. sawhney@rosewellpark.org
Rajinder S
RS
Sawhney
Michelle M
MM
Cookson
Bhavya
B
Sharma
Jennie
J
Hauser
Michael G
MG
Brattain
D019898:Autocrine Communication; D001665:Binding Sites; D019921:Biotinylation; D015153:Blotting, Western; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002465:Cell Movement; D003110:Colonic Neoplasms / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D005799:Genes, Dominant; D006801:Humans; D015151:Immunoblotting; D007211:Indoles / Q000494:pharmacology; D039421:Integrin alpha2 / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000378:metabolism / Q000502:physiology*
D004791:Enzyme Inhibitors; D007211:Indoles; D039421:Integrin alpha2; D008301:Maleimides; D016211:Transforming Growth Factor alpha; D013912:Threonine; D012694:Serine; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D011493:Protein Kinase C; D020929:Mitogen-Activated Protein Kinase Kinases; C088060:bisindolylmaleimide; D020123:Sirolimus