Activation of focal adhesion kinase in human lung cancer cells involves multiple and potentially parallel signaling events.
Integrins are adhesion receptors that transmit signals bidirectionally across the plasma membrane. In our previous report we have shown that the squamous lung cancer cell line, Calu-1, binds to collagen type IV (Coll IV) through beta1-integrin and results in phosphorylation of focal adhesion kinase (FAK) (Ann Thorac Surg 2004; 78:450-457). Considering the critical role of FAK in cell migration, proliferation, and survival, here we investigated potential mechanisms of its activation and regulation in Calu-1 cells. We observed the phosphorylation of Tyr397 of FAK (the autophosphorylation site of FAK) and paxillin, the immediate downstream substrate of FAK following the adhesion of Calu-1 cells to Coll IV. FAK remains phosphorylated during proliferation either on Coll IV or on uncoated plates for 72 h, as determined by peroxivanadate treatment. Exposure of Calu-1 cells with 60 microM genistein, reduces FAK phosphorylation (7.6 fold) and cell proliferation. Extracellular signal regulated kinases (ERKs) were also phosphorylated after Coll IV attachment. Disruption of Calu-1 cell cytoskeleton integrity by 1-5 muM Cytochalasin D resulted in the inhibition of cell adhesion (50% to 75%, p<0.19 - 6.6 x 10(7)) and ERKs phosphorylation (2 fold) without any effect on FAK phosphorylation. Protein Kinase C inhibitor, Calphostin C at 100 and 250 nM concentrations did not block Coll IV induced FAK phosphorylation but activated the ERKs in a dose dependent manner. beta1-integrin is essential for Coll IV induced FAK activation, but it is not physically associated with FAK as determined by immunodetection assay. Collectively, this report defines the existence of multiple and potentially parallel Coll IV/beta1-integrin mediated signaling events in Calu-1 cells, which involve FAK, ERKs, and PKC.
Mukhopadhyay
Nishit K
NK
Division of Thoracic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, 02115, USA.
Gordon
Gavin J
GJ
Chen
Chang-Jie
CJ
Bueno
Raphael
R
Sugarbaker
David J
DJ
Jaklitsch
Michael T
MT
D002448:Cell Adhesion / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D024141:Collagen Type IV / Q000378:metabolism; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism; D008175:Lung Neoplasms / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D009281:Naphthalenes / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism
D024141:Collagen Type IV; D004791:Enzyme Inhibitors; D019012:Integrin beta1; D009281:Naphthalenes; D015638:Cytochalasin D; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; C058819:calphostin C