Proteolytic cleavage and activation of PAK2 during UV irradiation-induced apoptosis in A431 cells

Abstract

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.

Rho GTPases and their effector proteins

“…In agreement with the model a peptide from the PAK1 regulatory region (amino acids 83-149) can be used as a dominant-negative inhibitor of PAK activity both in itro and in i o [82], and mutants of the PAK inhibitory domain are found to have constitutive kinase activity [82,83]. Equally, cleavage of PAK-2 (and of PKN, see below) by caspases, which occurs during apoptosis, also removes its regulatory domain and creates a constitutively active protein [84,85]. Two kinases which are Rho effectors have also been reported to contain autoinhibitory domains, ROK and PKN.…”

Huntingtin promotes cell survival by preventing Pak2 cleavage

“…Also, in contrast to Pak1 and Pak3, Pak2 can be activated via cleavage by caspase proteases to release a constitutively active C-terminal fragment (Pak2p34). Caspaseactivated Pak2p34 has been observed after cells are exposed to death stimuli such as Fas ligand, TNFα, heat shock or UV radiation Rudel and Bokoch, 1997;Tang et al, 1998). Studies have suggested that the formation of Pak2p34 mediates cell death in response to these stimuli and that it is an essential mediator of cell death in response to Fas-related signals (Rudel and Bokoch, 1997;Rudel et al, 1998).…”

A Novel Role for p21-Activated Protein Kinase 2 in T Cell Activation

“…In contrast, PAK2 has been reported to be activated by cellular stresses such as hyperosmolarity and DNA damage (19,20). In Jurkat T cells, PAK2 is activated after cleavage by caspase 3 and appears to mediate some of the membrane and morphological changes characteristic of programmed cell death (21).…”