In this work, we show that a group B Pak, Pak5, has properties that are distinct from all other Paks described to date. Pak5 is distinguished from group A Paks by its inability to complement STE20 function in budding yeast, by its high basal activity that is not regulated by GTPases, and by its marked preference for binding Cdc42 over Rac. While Pak5 shares some common properties with the group B enzyme Pak4, such as conferring protection from apoptotic stimuli and the ability to phosphorylate BAD on Ser-112, Pak5 is localized to mitochondria. These features suggest that Pak5 plays a different role than Pak4 in regulating apoptosis.
Consistent with the absence of apoptosis in cells expressing Pak5, we observed an absence of caspase 3 cleavage and the subsequent PARP cleavage, two key features of programmed cell death. In contrast, expression of a KD mutant of Pak5 does not protect against apoptosis, and does not prevent caspase 3 and PARP cleavage. Therefore, we conclude that Pak5 kinase activity is responsible for resistance to apoptosis. Interestingly, activation of the group A enzyme, Pak2, is associated with proapoptotic rather than antiapoptotic effects. One key difference between proapoptotic kinases such as Pak2 and antiapoptotic kinases such as Pak1, Pak4, and Pak5 is that the former contains a caspase site and is sensitive to cleavage by caspase 3 (26
), whereas the latter lack such sites and are not known to be cleaved. Possibly, the generation of a separated kinase domain, such as occurs following Pak2 cleavage, results in relocalization and acquisition of new substrates that contribute to cell death. Therefore, we suspect that the N terminal portion of Pak5 regulates the localization and substrate specificity of this kinase, and is required for its pro-survival effects.
One of the mechanisms by which Pak5 induces resistance to apoptosis is the phosphorylation of BAD. We show that Pak5 phosphorylates BAD in vitro and in vivo, as it has also been observed for Pak1 and Pak4 (18
). These phosphorylations occur at Ser-112, and perhaps, to a lesser extent, on additional Ser residues. While Pak5 may have additional targets that are relevant to its antiapoptotic effects, the phosphorylation of BAD is likely to be germane in this setting. Although Pak5 can phosphorylate BAD in vitro and induce BAD phosphorylation in cells, there are several other kinases that also exhibit these properties. To date, PKA has been identified as a major BAD Ser-112 kinase, while Akt has been reported to be a major Ser-136 kinase. Our data suggest that the Pak5-stimulated phosphorylation of BAD at Ser-112 is direct, as Pak5 acts on this site in vitro and inhibitors of PKA do not affect the ability of Pak5 to maintain BAD Ser-112 phosphorylation in cells treated with inducers of apoptosis. In contrast, the Pak5-stimulated phosphorylation of BAD at Ser-136 may be mediated through Akt, as Pak5 expression is associated with elevated activity of Akt. These results suggest that Pak5 and PKA are independent mediators of BAD phosphorylation (Fig. ). They also suggest that Pak5 signaling differs from Pak1 which appears to act downstream of Akt (38
Model of Pak5 in cell survival signaling.
The phosphorylation of BAD by Pak5 is likely to occur at the mitochondrion, as Pak5 appears to be a constitutive resident of this organelle, and BAD translocates to mitochondria following apoptotic stimuli. The predominantly mitochondrial localization of Pak5 is independent of cell type, as we were able to see that same colocalization of Pak5 in CHO, HMN1 and NIH-3T3 cells (data not shown). Pak5 is not recruited to mitochondria specifically during apoptosis, and its residence there is independent of kinase activity or its ability to bind Cdc42. In this respect, Pak5 resembles the mitochondrion-anchored PKA, although we do not know if Pak5 is anchored to the mitochondrion directly or bound to a mitochondrion-anchored protein.
To date, localization of other group B Pak isoforms has not been studied in detail; therefore, we also assessed Pak4 and Pak6 localization. Surprisingly, we found that Pak6, and, to a lesser extent, Pak4, also localized to mitochondria. The bulk of Pak4, however, is detected in the light microsome fraction (Fig. ), in agreement with the findings of Abo et al. (1
), who showed that Pak4 translocates to Golgi apparatus when coexpressed with Cdc42. Pak6 was previously shown to be cytosolic and to shuttle to the nucleus when coexpressed with androgen and estrogen receptors (27
), but has not been previously reported to localize to mitochondria. Our data suggest that mitochondrial localization is a common feature of group B Paks.
Mammalian group B Paks appear to localize to many distinct cellular compartments, unlike group A Paks such as Pak1, which are found mostly in areas of active actin dynamics, such as leading edge lamellipodia (37
) or the group B X-Pak5 from Xenopus
, which has been found in association with microtubules (5
). The mammalian group B Paks are in general less similar to one another than are the group A Paks, and it is likely that this diverged sequence reflects diverged cellular function. While we do not yet know what domain on Pak5 targets the mitochondrion, the N terminus is probably involved. Paks 4, 5, and 6 differ substantially in this noncatalytic N-terminal domain, whereas the C-terminal kinase domains are very similar. Moreover preliminary data from our lab shows that the isolated N terminus of Pak5 localizes to mitochondria. Whether the Cdc42 binding domain, which is located at the extreme N terminus of the protein, is involved is currently unknown but appears unlikely as Cdc42 is not known to localize to this organelle.
Pak5 expression is restricted to the brain and overexpression of this kinase has been shown to induce neurite outgrowth in N1E-115 cells (8
). It is likely that such kinase activity is tightly controlled in central nervous system, as 95% of neurons undergo apoptosis during embryogenesis. How is this kinase regulated? Pak5 has unusually high basal activity compared to other group A or group B Paks. While Pak5 binds Cdc42, this interaction has no effect on kinase activity in vitro. This finding suggests that the binding to Cdc42 could have a role other than direct activation. However, it is also possible that there are endogenous inhibitors of Pak5 that regulate its activity under physiological conditions. Provided that adequate sera can be raised to the endogenous protein, we plan to examine these and related issues in the near future.