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Fox Chase Cancer Center, Philadelphia, Pennsylvania
Received 6 December 2002/ Returned for modification 27 January 2003/ Accepted 15 May 2003
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ABSTRACT |
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INTRODUCTION |
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As effectors of Rho family GTPases, Paks play an important role in the regulation of morphology and motility by regulating the actin cytoskeleton (13, 15, 25, 29, 34, 36). While we know in reasonable detail how group A Paks are regulated and what their targets are, we know much less about the group B Paks. Group B Paks are much less closely related to one another than are group A Paks, especially in the regions outside the CRIB and kinase domains. Of the group B Paks, Pak4 is the best characterized. Unlike that of Paks 1, 2, and 3, the interaction of Cdc42 with Pak4 is thought to regulate translocation of this kinase to the Golgi apparatus, rather than its activation (1). Pak4 is involved in reorganization of the actin cytoskeleton, but only one likely substrate (LIM kinase) has been identified thus far (7). Pak6 interacts with the androgen and estrogen receptors and has been reported by one group to translocate into the nucleus with the androgen receptor in response to androgen (27, 41). Pak5 is expressed primarily in neuronal tissues and induces neurite-like projections when overexpressed in NIE-115 cells (8). These data suggest that the group B Paks have disparate cellular functions.
Both group A and B Paks have been implicated in apoptosis, possessing either proapoptotic (Pak2) or antiapoptotic (Pak1 and Pak4) properties (18, 23, 31, 32, 38). Pak2 is cleaved during apoptosis, most likely by caspase 3; this cleavage leads to its activation, and the activated kinases in turn are thought to contribute to morphological and membrane changes that occur during apoptosis (26, 31, 40). In contrast Pak1 and Pak4, which are not cleaved by caspases, have been reported to protect cells from apoptosis (18, 31, 32, 38). The survival signal induced by Paks is thought to be related to phosphorylation of the apoptotic protein BAD on Ser-112 (Pak1 and Pak4) and Ser-136 (Pak1) (18, 31, 32, 38), though it is not known if these phosphorylations are directly catalyzed by Paks in vivo. BAD is a proapoptotic member of the BCL-2 family that has been shown to translocate between cytosol and mitochondrial membrane-based partners such as BCL-2 or BCL-xL. This complex formation inhibits the ability of BCL-2 and BCL-xL to block the release of cytochrome c from mitochondria, a critical step in the activation of the downstream caspase protease cascade (16, 19, 20). BAD is rapidly phosphorylated on two serine residues in response to a survival factor, interleukin-3. Activated Akt has been shown to phosphorylate the proapoptotic BAD protein on Ser-136, while a recent study identified mitochondrion-associated protein kinase A (PKA) as a BAD Ser-112-specific kinase (17). The phosphorylation of BAD results in its dissociation from complexes with BCL-2 and BCL-xL and its subsequent association with the cytosolic adapter protein 14-3-3
(10, 11). The uncomplexed BCL-xL is then capable of suppressing cell death responses by blocking the release of mitochondrial cytochrome c (18).
Here, we investigate the biochemical and functional properties of Pak5, particularly with regard to its role in apoptosis. We show that Pak5 function differs from that of group A Paks such as Pak1, since it does not complement Ste20 and it is not activated by GTPases. We show that Pak5 induces resistance to apoptosis induced by two distinct stimuli: camptothecin (CA), a topoisomerase I inhibitor, and C2-ceramide (C2C), a well-known analog to a second messenger released from plasma membrane during apoptosis. Expression of Pak5 inhibits PARP and caspase 3 cleavage and promotes BAD phosphorylation on Ser-112, whereas the KD mutant does not protect against apoptosis. Pak5 phosphorylates BAD Ser-112 independently of PKA, and also promotes the phosphorylation of Ser-136, most likely via activation of Akt. Moreover, Pak5 prevents BAD translocation to mitochondria and more importantly, Pak5 localizes to mitochondria, and this localization is independent of kinase activity or the ability to bind Cdc42.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmids. Pak5 cDNA was obtained from the RIKEN cDNA collection and subcloned in pCMV6 containing a Myc epitope tag (36). Kinase-dead (KD) (K478M) mutant of Pak5 (8) was constructed by site-directed mutagenesis (Stratagene QuikChange kit). These Pak5 variants along with the constitutively active allele of Pak5 (PAK5 S573N) were cloned into the S. cerevisiae expression vector pYes2 (Invitrogen). Additionally human Pak2 cloned into pYES2 was also used as a positive control. Plasmids encoding GST-tagged and HA-tagged Cdc42 L61 and N17 and Rac1 L61 and N17 were constructed as described elsewhere (33). The Pak4-encoding vector was obtained from Audrey Minden (Columbia University, New York, N.Y.). pEBG-BAD encoding for GST-BAD was purchased from Cell Signaling. pET vectors encoding His-tagged BAD, BAD S112A, and BAD S136A and pcDNA-HA-BAD were a gift from Michael Greenberg (Harvard Medical School, Boston, Mass.). The BAD S112A/S136A double mutant was obtained by site-directed mutagenesis. HIS-BAD proteins were inducibly expressed in Escherichia coli strain BL21(DE3)pLysS and purified over Ni-nitrilotriacetic acid resin (Qiagen).
Complementation analysis.
An S. cerevisiae strain bearing a deletion in STE20 (MATa ade2 his3-
1 leu2-0 met15-0 ura3-0 ste20::KanMX) or the parental isogenic strain BY4741 (Research Genetics) was transformed by a lithium acetate procedure (17) with a galactose-inducible expression vector, pYES2 bearing either no insert or a cDNA encoding Pak-2, Pak5, Pak5 K478M (KD), or S573N (hyperactivated) mutants. Transformants were selected for growth on minimal medium lacking uracil. To perform qualitative mating assays, the transformants were serially diluted onto minimal raffinose plates lacking uracil (URA- plates). After the colonies had grown sufficiently the plates were replica plated onto either yeast nutrient broth (YNB) or Gal URA- plates and allowed to express the transgene for 5 h at 30°C. These plates were then replica plated onto YEPD plates that contained a lawn of the mating tester strain RSY16 (MAT
ade2-1 leu1-2 lys2-1 trp5-20 ura1) (a gift from R. Strich, Fox Chase Cancer Center), and the cells were allowed to mate for 6 h before being replicated plated onto GN plates (0.17% YNB, 2% glucose, 0.5% ammonium sulfate). Mating efficiency was scored by the formation of prototrophic strains on the GN plates.
Protein isolation from S. cerevisiae and immunoprecipitation kinase assays. Fifty-milliliter cultures of the transformants were grown in Raff URA- medium. Galactose was added to 2% and the cells were allowed to express the transgene for 5 h. At time zero and 5 h, 25-ml fractions were collected by centrifugation and frozen on dry ice. The pellets were resuspended in 1.0 ml of Tris-EDTA and divided as follows: 100 µl was transferred to an Eppendorf tube, collected by centrifugation, and lysed by boiling in 1x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer for 10 min. The remaining 900 µl was collected by centrifugation and lysed with YPER buffer (Pierce) (5 ml/g of cell pellet) for 25 min at room temperature. The lysates were centrifuged, and the supernatant was transferred to an Eppendorf tube. The remaining pellet was resuspended in 500 µl of radioimmunoprecipitation (RIPA) buffer and subjected to four rounds of freeze-thaw in liquid nitrogen. The lysates were centrifuged for 10 min at 14,000 rpm, and the supernatant was transferred to the tube containing the YPER lysate supernatant. The combined lysates were used for immunoprecipitation using the anti-Myc polyclonal antibody A14 or the anti-Pak2 specific antibody overnight at 4°C. Protein A/G was added for 2 h and the immunoprecipitates were washed five times with lysis buffer (66% YPER buffer-33% RIPA buffer), then three times with kinase assay buffer. Although the YPER-RIPA lysis was effective, we could not immunologically detect expression of the transgenes in the lysates or in the immunoprecipitations suggesting a problem of limited solubility of the mammalian Paks in S. cerevisiae. Multiple extraction procedures failed to resolve this limitation. Kinase assays were performed as described below.
Cell culture, transfection and treatment. All cell lines were grown at 37°C in 5% CO2 and cultured in Dulbecco's modified Eagle's medium for COS cells and Dulbecco's modified Eagle's medium-F-12 for CHO cells containing 10% fetal bovine serum, and puromycin (2 µg/ml; Sigma) for CHO stable cell lines transfected with either empty vector (pCMV6, control cells), wild-type (WT) Pak5, or KD Pak5 K478M. HMN1 cells were kindly provided by R. Pittman (University of Pennsylvania, Philadelphia). Cells were transfected using Lipofectamine (Gibco), and lysates were collected 48 h after transfection. CHO stable cell lines transfected with pcDNA-HA-BAD were treated 24 h after transfection with CA (10 µM) or C2C (50 µM).
GST pull down assay.
To perform Rac and Cdc42 binding assays, 50 µg of purified GST fusion proteins coupled to glutathione-Sepharose beads (Amersham Pharmacia Biotech) were incubated with buffer A (40 mM HEPES [pH 7.4], NP-40 1%, 1 mM EDTA, 150 mM NaCl) supplemented with 10 mM EDTA for 15 min at room temperature to release any nucleotide, washed with buffer A, incubated in buffer B (buffer A supplemented with 1 mM GTP-
S and 10 mM MgCl2) for 30 min at room temperature, and then washed with buffer B. Next, the proteins were incubated with 250 µl of Cos lysates for 3 h at 4°C. Precipitates were washed three times with lysis buffer (described below). Bound proteins were eluted in SDS sample buffer and subjected to immunoblotting with anti-Myc monoclonal antibody 9E10.
Immunoblot and immunoprecipitation. Cos cells were transfected with appropriate vectors. Cell extracts were obtained in lysis buffer (50 mM Tris [pH 8], 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 0.5 mM phenylmethylsulfonyl fluoride, aprotinin [1 µg/ml]), protein concentration was assessed using bicinchoninic acid (Pierce) and equal amounts of proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Immobilon P; Millipore Corp.). Blots were blocked 1 h in Tris-buffered saline-0.1% Tween 20 supplemented with 5% nonfat milk, and incubated with primary antibodies for an hour or overnight at 4°C. After washing, blots were incubated with alkaline phosphatase-conjugated secondary antibodies (Jackson Laboratories), which were detected using the CPD Star reagent (New England Nuclear). The immunoprecipitation assay was carried out as follows: whole-cell lysates were incubated with polyclonal anti-Myc A14 overnight at 4°C and then 1 h at 4°C with protein A agarose beads (Pierce). Beads were collected by centrifugation and washed with the lysis buffer described above. Proteins were eluted by boiling in SDS sample buffer and subjected to immunoblotting, and probed with anti-HA (12CA5J).
Protein kinase assay.
Cos cells were transfected with appropriate expression vectors. Transfected cells were collected in lysis buffer 48 h after transfection and equal amounts of the Myc-tagged proteins, as assayed by Western blotting, were then immunoprecipitated with anti-Myc antibody A14 and protein A-agarose. After incubation, the immunoprecipitates were washed twice in lysis buffer and twice in a kinase buffer (50 mM Tris [pH 7.5], 100 mM NaCl, 1 mM MnCl2, 10 mM MgCl2, 1 mM dithiothreitol) and then were incubated with 1.5 µg of MBP or His-tagged BAD recombinant proteins in the kinase buffer supplemented with 20 µM ATP and 5 µCi of [-32P]ATP for 30 min at 30°C. The reaction was terminated with SDS-PAGE sample buffer, followed by SDS-PAGE and autoradiography.
Apoptosis assays. To estimate apoptosis of stably transfected cell lines, equal numbers of cells were seeded in six-well plates. Cells were treated 16 h with CA (10 µM) or C2C (50 µM), and collected for flow cytometry or used to prepare lysates. Cells were collected by trypsinization and, after combination with the floating cells, were labeled with annexin V and propidium iodide according to the manufacturer's recommendations (Clontech) for fluorescence-activated cell sorting analysis (Becton Dickinson). When the PKA inhibitor (PKI) was used, cells were pretreated 30 min with 1 µM before inducers were added for 16 h.
Cellular fractionation. CHO cell lines were transfected with pcDNA-HA-BAD and after 24 h were treated for 16 h with CA (10 µM) or C2C (50 µM). Cellular fractionation was then performed as previously described (14). Briefly, cells were trypsinized and resuspended in isotonic mitochondrial buffer (MB) (210 mM mannitol, 70 mM sucrose, 1 mM EDTA, 10 mM HEPES [pH 7.5] supplemented with proteases inhibitors). Cells were broken by passages through a 25-gauge needle fitted on a syringe, and the suspension was centrifuged at 2,000 x g at 4°C. Supernatants containing heavy membranes were subjected to a 13,000 x g centrifugation at 4°C, the supernatant corresponding to the cytosolic fraction was centrifuged at 100,000 x g for 30 min, and the pellet containing mitochondria was resuspended in MB and submitted to a 500 x g centrifugation. The resultant supernatant was centrifuged 10 min at 10,000 x g to collect the mitochondrial fraction. A further purification step was performed for Pak's subcellular localization (24). The latter mitochondrial fraction was resuspended in MB and applied on a 1-to-2 M linear sucrose gradient and centrifuged 30 min at 60,000 x g using an SW28 Beckman rotor. The mitochondria, which banded at a region corresponding to 1.5 M were collected and diluted in MB to 0.25 M and pelleted at 15,000 x g for 15 min. The mitochondrial pellets were resuspended in MB. The S100 cytosolic fraction also generated the light microsomal pellet after centrifugation, which was resuspended in buffer H (20 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 6 mM ß-mercaptoethanol [pH 7.5], 1% SDS).
Immunofluorescence. HMN1 cells seeded on coverslips were transiently transfected with empty vector or vectors encoding Myc-tagged WT Pak5 or KD Pak5 (K478M). HMN1 and CHO cells were stained 15 min at 37°C with a 5-ng/ml concentration of the mitochondrion-specific dye MitoTracker Red CMXRos (Molecular Probes Inc.) 24 h after transfection and then fixed in 4% paraformaldehyde. Cells were fixed, permeabilized in PBS-0.1% NP-40, and blocked 30 min with 10% bovine serum albumin. Cells were stained for the presence of Pak5 with rabbit polyclonal anti-Myc antibody (A14) and incubated with anti-rabbit immunoglobulin G conjugated with fluorescein isothiocyanate (FITC) (Jackson Laboratories). Cells were then washed, mounted and photomicrographs were obtained using a Bio-Rad radiant 2000 laser scanning confocal microscope with a Plan APO 60x water immersion objective (1.20 numerical aperture; Nikon E800 Eclipse).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The group A Paks requires binding of GTPases to be activated whereas Pak4 has been shown in vitro to autophosphorylate and to phosphorylate Histone H4 (5). We assessed Pak5 kinase activity using MBP as a substrate (Fig. 2C). COS cells were transfected with Myc-tagged Pak5 expression vector or Myc-tagged Pak1, used as a positive control. GTPases expression vectors were also cotransfected. Equal amounts of Pak5 and Pak1 were immunopurified from cell lysates and incubated with MBP and [-32P]ATP in kinase buffer. Autophosphorylation of Pak5 and transphosphorylation of MBP in Fig. 2C shows that Pak5 has a strong autophosphorylation activity compared to Pak1 and phosphorylates MBP in the absence of GTPases. On the contrary, Pak1 requires activation by GTPases in order to phosphorylate MBP, and does so at orders of magnitude less than Pak5 (Fig. 2C, inset).
Pak5 induces resistance to apoptosis induced by ceramide or CA. As many of the Paks have previously been shown to either induce (Pak2, once cleaved by caspase 3) (26, 31, 40) or inhibit (Pak1 and Pak4) (18, 32, 38) apoptosis, we sought to determine if Pak5 also plays a role in this process. In order to examine the role of Pak5 in cell survival, CHO stable cell lines were generated expressing either WT Pak5 or KD Pak5 (K478M) (8) or empty vector (control). At least three independent stable clones were analyzed for each construct. Representative data for protein expression and kinase activity data are shown (Fig. 3A). Consistent with the data from transiently transfected COS cells, WT Pak5 in CHO cells exhibits considerable basal activity, whereas the K478 M mutant is inactive.
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We examined PARP and caspase 3 cleavage, two biochemical markers of apoptosis, in the stable cell lines. After a 16-h treatment with C2C and CA, PARP and caspase 3 were cleaved in control and KD Pak5 cell lines (Fig. 3C and D), whereas WT Pak5-expressing cell lines, which are resistant to apoptosis, retain full-length PARP and caspase 3 in both untreated and induced cells. This result indicates that the apoptotic signal leading to the release of cytochrome c from the mitochondrion, subsequently activating caspase 9 and caspase 3, is blocked downstream of the mitochondrion when Pak5 is expressed.
Pak5 phosphorylates BAD. Activated Pak1 and Pak4 have been shown to phosphorylate BAD on Ser-112 and Ser-136 and Ser-112, respectively (18, 32). We addressed whether BAD is a target for Pak5, BAD being a key proapoptotic molecule. To analyze BAD phosphorylation by Pak5, COS cells were transfected with empty vector, WT Pak5, KD Pak5 or WT Pak4 as a positive control. In vitro kinase assay was carried out in which His-tagged recombinant WT BAD, S112A or S136A mutants or double mutant S112A/S136A proteins were used as substrates for immunopurified Pak proteins (Fig. 4A). These results indicate that WT Pak5 and Pak4 cannot phosphorylate BAD S112A mutant or the double mutant S112A/S136A, whereas they are able to phosphorylate WT BAD or the S136A mutant. Therefore, Pak5 phosphorylates BAD mainly on Ser-112, although BAD mutation on Ser-112 does not completely abolish its phosphorylation, indicating that Pak5 could target to a lesser extent additional residues. In order to confirm this result, we used phosphospecific antibodies directed against Ser-112 or Ser-136 to determine the status of the 112 and 136 sites in an in vitro non radioactive kinase assay (Fig. 4B). These data show that Pak5 phosphorylates BAD on Ser-112, in agreement with the radioactive kinase activity assay.
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Pak5 prevents BAD translocation to mitochondria.
Under normal condition, phosphorylated BAD is sequestered by 14-3-3 in the cytosol (42). In response to apoptotic stimuli, BAD is dephosphorylated and translocates to the mitochondrial membrane where it binds to the antiapoptotic proteins BCL-2 and BCL-xL (18). Having demonstrated that Pak5 phosphorylates BAD on serine 112 and indirectly on serine 136, we determined if this activity of Pak5 was sufficient to maintain BAD in the cytosol and particularly if it prevents BAD translocation to mitochondria, and therefore the apoptotic cascade. We transfected control, WT Pak5, and KD Pak5 stable cell lines with a pcDNA-BAD plasmid, and treated cells with inducers, CA or C2C, in order to induce apoptosis, and subsequent BAD translocation to mitochondria. We found that BAD translocates to mitochondria in both control and KD Pak5 CHO cells, as shown by the presence of BAD in the mitochondrial fraction, whether apoptosis is induced by CA (Fig. 7A) or C2C (Fig. 7B). In contrast, no translocation occurs in the WT Pak5 cell lines, as suggested by the absence of BAD in the mitochondrial fraction of these cells. This is consistent with the phosphorylation of serine 112 by Pak5 and shows that Pak5, by its phosphorylation activity, can prevent BAD translocation to mitochondria.
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We next studied the localization of other group B Paks in order to determine if either of these could also localize to mitochondria. We transfected CHO cell lines with vectors encoding HA-Pak4 and HA-Pak6. The results show that Pak4 is predominantly localized in the light microsome fraction, and a smaller fraction is also found in the mitochondrial fraction (Fig. 8C). Surprisingly, Pak6, like Pak5, was found exclusively in the mitochondrial fraction.
To confirm the mitochondrial localization of Pak5, immunofluorescent staining was performed on the CHO stable cell lines. As shown in Fig. 9A, there is substantial colocalization of Myc-WT Pak5 with mitochondria, as assessed using MitoTracker. Similar results were obtained when other mitochondrial markers, such as HSP60 and COX-IV, were examined in Pak5-transfected cells (data not shown). As in the cell fractionation studies, kinase activity is not required for this colocalization, as a substantial proportion of the KD mutant also localized to mitochondria. We also tested in CHO cells the localization of a mutant of Pak5 that bears two point mutations (K19L and K23L) in the CRIB domain where Cdc42 binds (1, 31). These mutations do not affect Pak5 mitochondrial localization (data not shown).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, 31), 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, 31, 32, 38). 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. 10). They also suggest that Pak5 signaling differs from Pak1 which appears to act downstream of Akt (38).
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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. 8C), 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.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the National Institutes of Health (RO1 GM-54168 and CA-09035-27) and the American Cancer Society (CB-189), as well as CORE Grant CA-06927 and an appropriation from the Commonwealth of Pennsylvania.
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FOOTNOTES |
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