Integrin alpha2-mediated ERK and calpain activation play a critical role in cell adhesion and motility via focal adhesion kinase signaling: identification of a novel signaling pathway.
Higher levels of focal adhesion kinase (FAK) are expressed in colon metastatic carcinomas. However, the signaling pathways and their mechanisms that control cell adhesion and motility, important components of cancer metastasis, are not well understood. We sought to identify the integrin-mediated mechanism of FAK cleavage and downstream signaling as well as its role in motility in human colon cancer GEO cells. Our results demonstrate that phosphorylated FAK (tyrosine 397) is cleaved at distinct sites by integrin signaling when cells attach to collagen IV. Specific blocking antibodies (clone P1E6) to integrin alpha2 inhibited FAK activation and cell motility (micromotion). Ectopic expression of the FAK C-terminal domain FRNK attenuated FAK and ERK phosphorylation and micromotion. Calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal blocked FAK cleavage, cell adhesion, and micromotion. Antisense approaches established an important role for mu-calpain in cell motility. Expression of wild type mu-calpain increased cell micromotion, whereas its point mutant reversed the effect. Further, cytochalasin D inhibited FAK phosphorylation and cleavage, cell adhesion, locomotion, and ERK phosphorylation, thus showing FAK activation downstream of actin assembly. We also found a pivotal role for FAK Tyr(861) phosphorylation in cell motility and ERK activation. Our results reveal a novel functional connection between integrin alpha2 engagement, FAK, ERK, and mu-calpain activation in cell motility and a direct link between FAK cleavage and enhanced cell motility. The data suggest that blocking the integrin alpha2/FAK/ERK/mu-calpain pathway may be an important strategy to reduce cancer progression.
Sawhney
Rajinder S
RS
Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA. rajinder.sawhney@roswellpark.org
Cookson
Michelle M
MM
Omar
Yasin
Y
Hauser
Jennie
J
Brattain
Michael G
MG
D019921:Biotinylation; D002070:Butadienes / Q000494:pharmacology; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D003110:Colonic Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D015638:Cytochalasin D / Q000494:pharmacology; D017097:Electric Impedance; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D039421:Integrin alpha2 / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000494:pharmacology; D010766:Phosphorylation; D017354:Point Mutation; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism
D002070:Butadienes; D004791:Enzyme Inhibitors; D039421:Integrin alpha2; D009570:Nitriles; D019384:Nucleic Acid Synthesis Inhibitors; D016376:Oligonucleotides, Antisense; C113580:U 0126; D015638:Cytochalasin D; D014443:Tyrosine; D051416:Focal Adhesion Protein-Tyrosine Kinases; D020928:Mitogen-Activated Protein Kinases; D002154:Calpain; C449002:mu-calpain