Caspase-mediated cleavage of p130cas in etoposide-induced apoptotic Rat-1 cells.
Apoptosis causes characteristic morphological changes in cells, including membrane blebbing, cell detachment from the extracellular matrix, and loss of cell-cell contacts. We investigated the changes in focal adhesion proteins during etoposide-induced apoptosis in Rat-1 cells and found that during apoptosis, p130cas (Crk-associated substrate [Cas]) is cleaved by caspase-3. Sequence analysis showed that Cas contains 10 DXXD consensus sites preferred by caspase-3. We identified two of these sites (DVPD(416)G and DSPD(748)G) in vitro, and point mutations substituting the Asp of DVPD(416)G and DSPD(748)G with Glu blocked caspase-3-mediated cleavage. Cleavage at DVPD(416)G generated a 74-kDa fragment, which was in turn cleaved at DSPD(748)G, yielding 47- and 31-kDa fragments. Immunofluorescence microscopy revealed well-developed focal adhesion sites in control cells that dramatically declined in number in etoposide-treated cells. Cas cleavage correlated temporally with the onset of apoptosis and coincided with the loss of p125FAK (focal adhesion kinase [FAK]) from focal adhesion sites and the attenuation of Cas-paxillin interactions. Considering that Cas associates with FAK, paxillin, and other molecules involved in the integrin signaling pathway, these results suggest that caspase-mediated cleavage of Cas contributes to the disassembly of focal adhesion complexes and interrupts survival signals from the extracellular matrix.
Kook
S
S
Department of Life Science, Kwangju Institute of Science and Technology, Kwangju 500-712, Korea.
Shim
S R
SR
Choi
S J
SJ
Ahnn
J
J
Kim
J I
JI
Eom
S H
SH
Jung
Y K
YK
Paik
S G
SG
Song
W K
WK
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D020134:Catalytic Domain; D002448:Cell Adhesion / Q000187:drug effects; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002478:Cells, Cultured; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005047:Etoposide / Q000494:pharmacology*; D005109:Extracellular Matrix / Q000502:physiology; D016023:Integrins / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D011506:Proteins; D051381:Rats; D050717:Retinoblastoma-Like Protein p130
D000972:Antineoplastic Agents, Phytogenic; C493843:Bcar1 protein, rat; D015815:Cell Adhesion Molecules; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins; D016023:Integrins; D051419:Paxillin; D010750:Phosphoproteins; D011506:Proteins; C496063:Pxn protein, rat; D050717:Retinoblastoma-Like Protein p130; C108082:focal adhesion protein, 52-kDa; D005047:Etoposide; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases