Proteolytic cleavage and activation of PAK2 during UV irradiation-induced apoptosis in A431 cells.
Exposure of mammalian cells to ultraviolet (UV) light elicits a cellular response and can also lead to apoptotic cell death. In this report, we show that a 36-kDa myelin basic protein (MBP) kinase detected by an in-gel kinase assay can be dramatically activated during the early stages of UV irradiation-triggered apoptosis of A431 cells. Immunoblot analysis revealed that this 36-kDa MBP kinase could be recognized by an antibody against the C-terminal regions of a family of p21Cdc42/Rac-activated kinases (PAKs). By using this antibody and a PAK2-specific antibody against the N-terminal region of PAK2 as studying tools, we further demonstrated that UV irradiation caused cleavage of PAK2 to generate a 36-kDa C-terminal catalytic fragment and a 30-kDa N-terminal fragment in A431 cells. The appearance of the 36-kDa C-terminal catalytic fragment of PAK2 matched exactly with the activation of the 36-kDa MBP kinase in A431 cells upon UV irradiation. In addition, UV irradiation also led to activation of CPP32/caspase-3, but not ICH-1L/caspase-2 and ICE/caspase-1, in A431 cells and the kinetics of activation of CPP32/caspase-3 appeared to correlate well with that of DNA fragmentation and of cleavage/activation of PAK2, respectively. Moreover, blockage of activation of CPP32/caspase-3 by pretreating the cells with two specific tetrapeptidic inhibitors for caspases (Ac-DEVD-cho and Ac-YVAD-cmk) could significantly attenuate the extent of cleavage/activation of PAK2 induced by UV irradiation. Collectively, the results demonstrate that cleavage and activation of PAK2 can be induced during the early stages of UV irradiation-triggered apoptosis and indicate the involvement of CPP32/caspase-3 in this process.
Tang
T K
TK
Institute of Biochemical Sciences, National Taiwan University, Taipei, Republic of China.
Chang
W C
WC
Chan
W H
WH
Yang
S D
SD
Ni
M H
MH
Yu
J S
JS
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D017209:Apoptosis / Q000528:radiation effects*; D053148:Caspase 3; D020169:Caspases; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000276:immunology / Q000378:metabolism*; D014466:Ultraviolet Rays; D054462:p21-Activated Kinases
D000906:Antibodies; C515907:PAK2 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases