Caspase-activated PAK-2 Is Regulated by Subcellular Targeting and Proteasomal Degradation

Abstract

p21-activated protein kinases (PAKs) are a family of serine/threonine protein kinases that are activated by binding of the p21 G proteins Cdc42 or Rac. The ubiquitous PAK-2 (␥-PAK) is unique among the PAK isoforms because it is also activated through proteolytic cleavage by caspases or caspase-like proteases. In response to stress stimulants such as tumor necrosis factor ␣ or growth factor withdrawal, PAK-2 is activated as a fulllength enzyme and as a proteolytic PAK-2p34 fragment. Activation of full-length PAK-2 stimulates cell survival, whereas proteolytic activation of PAK-2p34 is involved in programmed cell death. Here we provide evidence that the proapoptotic effect of PAK-2p34 is regulated by subcellular targeting and degradation by the proteasome. Full-length PAK-2 is localized in the cytoplasm, whereas the proteolytic PAK-2p34 fragment translocates to the nucleus. Subcellular localization of PAK-2 is regulated by nuclear localization and nuclear export signal motifs. A nuclear export signal motif within the regulatory domain prevents nuclear localization of fulllength PAK-2. Proteolytic activation removes most of the regulatory domain and disrupts the nuclear export signal. The activated PAK-2p34 fragment contains a nuclear localization signal and translocates to the nucleus. However, levels of activated PAK-2p34 are tightly regulated through ubiquitination and degradation by the proteasome. Inhibition of degradation by blocking polyubiquitination results in significantly increased levels of PAK-2p34 and as a consequence, in stimulation of programmed cell death. Therefore, nuclear targeting and inhibition of degradation appear to be critical for stimulation of the cell death response by PAK-2p34.

Posttranslational myristoylation of caspase-activated p21-activated protein kinase 2 (PAK2) potentiates late apoptotic events

“…2 A). Contrasting with the nuclear localization of N-terminally tagged GFP-PAK2 constructs reported by Koeppel et al (24) and Jakobi et al (16), both of our C-t-PAK2-myc chimeras were excluded from the nucleus (Fig. 2 A).…”

“…Caspase-3-mediated cleavage of PAK2 has been observed during apoptosis (15,25,26), and overexpression of the Nterminally tagged C-terminal cleavage product C-t-PAK2 has been shown to induce cell death (16,24,27). However, all these previous studies assessed the apoptotic activity of the nonphysiologically relevant, nonmyristoylated form of C-t-PAK2.…”

“…Of the six PAK family members, only PAK2 is ubiquitously expressed and cleaved by caspase-3 (14)(15)(16). This cleavage removes the 28-kDa N-terminal regulatory domain and generates a constitutively active 34-kDa PAK2 C-terminal kinase fragment (C-t-PAK2) (15).…”

Unique and overlapping substrate specificities of caspase-8 and caspase-10

“…Cleavage of PAK2 at SHVD (212) separates the N-terminal regulatory from the C-terminal catalytic domain (Rudel and Bokoch, 1997;Lee et al, 1997), resulting in a constitutively active form of the kinase. Caspase cleavage, in addition, inactivates a nuclear export signal (NES, aa 197-246) resulting in nuclear accumulation of the fragment containing the catalytic domain and nuclear localization motif (NLS, aa 245-251) (Jacobi et al, 2003). Cleavage of CaMKLK (third panel) by caspases at DEND (62) leads to formation of an N-terminal proapoptotic p10 fragment and a C-terminal fragment with reduced kinase activity (Kruidering et al, 2001).…”

Coupling caspase cleavage and ubiquitin–proteasome-dependent degradation of SSRP1 during apoptosis

“…35 This coupling of degradation pathways could serve several functions including regulation of apoptotic cell death. Since many proteins involved in apoptosis are activated by caspase cleavage, 35 degradation of the resulting pro-or antiapoptotic fragments by the ubiquitin-proteasome pathway may add an additional level of regulation for decision making between survival and cell death. For instance, apoptotic cleavage might not completely inhibit SSRP1 activity as the N-terminal fragment probably still forms with Spt16 a chromatin-bound, truncated FACT complex.…”