Integrin α2-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

Rajinder S Sawhney,Michelle M Cookson,Yasin Omar,Jennie Hauser,Michael G Brattain

doi:10.1074/jbc.m600787200

Rajinder S Sawhney, Michelle M Cookson + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m600787200

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Abstract

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.

Highlights

Actin as well as other cytoskeletal proteins
GEO Cells Attach to Collagen type IV (CN IV) in a Concentration-dependent Fashion—There are several reports that integrin ␣2 is a receptor of CN I in different cell types [42, 43]
These results show that the ligand for integrin ␣2 receptor is cell type-specific

Summary

EXPERIMENTAL PROCEDURES

Materials—Methylthiazole tetrazolium (MTT), CN IV, Me2SO, BSA, cytochalasin D, soy bean trypsin inhibitor, and polyclonal antiactin antibody were purchased from Sigma. Cell lysates were incubated with streptavidin agarose for 90 min at 4 °C, and beads were washed five times with lysis buffer. Attachment and movement of the cells on the electrode change the flow of the current, resulting in fluctuations in the electrode resistance and capacitance. These cellular movements are called micromotion [39] and are a measure of the motile ability of the cell being measured. Since the measurements are electrical, they are quantitative and generate data that can be analyzed readily to provide sensitive measurements of changes in cell behavior This technique detects both translational (xy plane) and vertical (z direction) movement of cells [39]

RESULTS

DISCUSSION

Brattain