The Prostate-derived Sterile 20-like Kinase (PSK) Regulates Microtubule Organization and Stability*

Sterile 20 (STE20) protein kinases, which include germinal center kinases and p21-activated protein kinases, are known to activate mitogen-activated protein kinase pathways (c-Jun NH2-terminal kinase, p38, or extracellular signal-regulated kinase), leading to changes in gene transcription. Some STE20s can also regulate the cytoskeleton, and we have shown that the germinal center kinase-like kinase prostate-derived STE20-like kinase (PSK) affects actin cytoskeletal organization. Here, we demonstrate that PSK colocalizes with microtubules; and that this localization is disrupted by the microtubule depolymerizing agent nocodazole. The association of PSK with microtubules results in the production of stabilized perinuclear microtubule cables that are nocodazole-resistant and contain increased levels of acetylated α-tubulin. Kinase-defective PSK (K57A) or the C terminus of PSK (amino acids 745–1235) lacking the kinase domain are sufficient for microtubule binding and stabilization, demonstrating that the catalytic activity of the protein is not required. The localization of PSK to microtubules occurs via its C terminus, and PSK binds and phosphorylates α- and β-tubulin in vitro. The N terminus of PSK (1–940) is unable to bind or stabilize microtubules, demonstrating that PSK must associate with microtubules for their reorganization to occur. These results demonstrate that PSK interacts with microtubules and affects their organization and stability independently of PSK kinase activity.

nases have been identified, and these proteins divide into two subfamilies according to their structure and regulation (reviewed in Ref. Recent work has demonstrated that some STE20s can also regulate the cell cytoskeleton. PAKs interact with Rac or Cdc42 GTPases via the CRIB domain and act as downstream effectors for these small GTP binding proteins to regulate the actin cytoskeleton (reviewed in Refs. 4,5). Rac and Cdc42 stimulated morphological rearrangements are blocked by mutated PAK, and activated PAK down-regulates actin stress fibers and focal complexes (6 -8). Some of the effects of PAK1 can be attributed to the phosphorylation of downstream kinases, such as myosin light chain kinase, which reduces its activity toward myosin light chain, and LIM kinase 1, which phosphorylates and inactivates the actin depolymerizing protein cofilin to generate actin clusters (9,10). X-PAK5 co-localizes to actin and microtubules (MTs) and produces stabilized MTs that are associated in bundles, and PAK1 accumulates at the MT-organizing center and along mitotic spindles during mitosis (11,12).
Although GCK-like STE20s lack a CRIB domain, recent work has demonstrated that members of this subfamily of STE20s can also regulate the actin cytoskeleton. Proline-and alanine-rich STE20-related kinase associates with actin, and prostate-derived STE20-like kinase (PSK), Traf2-and Nckinteracting kinase, and STE20-like kinase (SLK) decrease actin stress fibers and focal adhesions and inhibit cell spreading (3,(13)(14)(15). SLK has recently been shown to associate with MTs at the cell periphery (16).
There is increasing evidence that actin filaments and MTs are coordinately regulated during the establishment and maintenance of cell polarity, division, and motility. The ability of MTs to undergo transitions between growth, shrinkage and pause are crucial for their function and the regulation of these processes. MT dynamics and stability are controlled by multiple factors, which include MT-associated proteins (MAPs), MTaffinity regulated kinases, severing factors (e.g. katanin), and catastrophe proteins (e.g. stathmin/OP18 and XKCM1) (reviewed in (17)). MAPs provide a focal point for MT regulation and act as structural proteins that lack enzymatic activity but promote the assembly of tubulin and MT stability. The affinity of MAPs for MTs is controlled by their phosphorylation, which causes the detachment of MAPs from MTs and results in MT * This research was supported by the Biotechnology and Biological Sciences Research Council, UK (Grant 29/C14086), the Association for International Cancer Research, St. Andrews, Scotland, the Ludwig Institute for Cancer Research, and by a donation from Laura Price. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Here we show that a recently identified member of the GCKlike family of STE20s, PSK (3), colocalizes with MTs and produces stabilized perinuclear MT cables that are nocodazoleresistant and contain increased levels of acetylated ␣-tubulin. These findings suggest that a major function for PSK is to transduce signals to the MT network.
Microinjection-Cells were grown in 10% fetal calf serum/Dulbecco's modified Eagle's medium. For microinjection, cells were seeded on glass coverslips and incubated for 2 days. Where appropriate, cells were serum-starved for 16 h before microinjection. Endotoxin-free plasmid DNA (100 ng/l in phosphate-buffered saline; Qiagen) was microinjected into the nuclei of ϳ100 cells per coverslip. After 3-4 h, cultures were fixed for 20 min with 4% paraformaldehyde, 10% Me 2 SO, and 1% glucose in phosphate-buffered saline (37°C) and permeabilized with 0.2% Triton X-100 for 5 min.
Immunoprecipitation-Cells were lysed as described above, and 400 g of protein were incubated with rabbit anti-PSK serum for 1 h at 4°C. Protein A-Sepharose beads (Sigma) were added to each sample, and after 1 h, beads were pelleted by centrifugation and washed three times in lysis and binding buffer. ter 60 min at 37°C, kinase assays were terminated in gel sample buffer and processed as described previously (3).

RESULTS
PSK Localizes to Microtubules-We have reported previously that PSK, a GCK-like kinase, activates the JNK MAPK pathway and regulates the actin cytoskeleton, and it showed a punctate or filamentous distribution when expressed in fibroblasts (3). To investigate the subcellular distribution of PSK in more detail, expression vectors encoding MYC-epitope tagged PSK were microinjected into the nuclei of Swiss 3T3 cells, and PSK was found to localize to a filamentous network in the cytoplasm of injected cells that closely resembled MTs (Fig.  1A). The localization of PSK to MTs was confirmed by costain-ing injected cells for PSK and the MT component ␤-tubulin (Fig. 1B). The immunofluorescence from PSK was more intense from MTs positioned around the nucleus than from MTs at the periphery of the cell, which appeared fragmented (Fig. 1, A and  B). The staining patterns for both PSK and ␤-tubulin were disrupted by addition of the MT-depolymerizing agent nocodazole (5 M), demonstrating that PSK and ␤-tubulin are closely associated (Fig. 1, C and D). Moreover, the fixation of cells in formaldehyde, not paraformaldehyde/Me 2 SO, resulted in the disruption of MTs and a tubulovesicular staining pattern for PSK (3). Costaining of injected cells for PSK and markers for other cytoskeletal structures (vimentin and actin) demonstrated that PSK localized specifically to the MT network (data not shown).
PSK Localizes to MTs via its C Terminus and Alters MT Organization-To determine whether the catalytic activity and/or specific sequences outside of the kinase domain of PSK are involved in localizing PSK to MTs, we prepared expression vectors encoding MYC-tagged full-length kinase-defective PSK (K57A) and the C terminus of PSK (amino acids 745-1235) for comparison with wild-type PSK and the N terminus of PSK (amino acids 1-940) containing the kinase domain (Fig. 2). PSK and kinase-defective PSK (K57A) both colocalized with ␤-tubulin (Fig. 3, A-D) showing that the catalytic activity of the protein is not required for its localization. PSK (745-1235), which lacks the entire kinase domain, also localized to MTs (Fig. 3, E and F), whereas PSK (1-940), containing the kinase domain, failed to localize to MTs and was cytoplasmic (Fig. 3, G  and H). Taken together, these results demonstrate that the C terminus of PSK is both necessary and sufficient for PSK to localize to the MT network, and this process occurs independently of the protein's catalytic activity.
The localization of PSK to MTs resulted in a significant change in MT organization. PSK generated MT cables around the nucleus that were absent in uninjected surrounding cells (Fig. 3, A and B). PSK (K57A) and PSK (745-1235), which both localize to MTs, also produced perinuclear MT cables (Fig. 3, C-F), and PSK was predominantly localized on these structures. Moreover, MTs appeared to be thicker in the presence of PSK, and co-staining for injected PSK and pericentrin, a marker for the MT-organizing center, demonstrated that the MT-organizing center was retained in cells expressing PSK (data not shown). PSK (1-940), which is catalytically active, failed to produce these MT structures, demonstrating that PSK must directly associate with MTs via its C terminus for their reorganization to occur (Fig. 3, G and H).
PSK Stabilizes MTs and Increases Acetylation of ␣-Tubulin-The ability of PSK to reorganize MTs into perinuclear cables raised the possibility that PSK might also alter the stability of MTs. To determine whether PSK could affect MT  4, A and B). The two other forms of PSK that localize to MTs, kinase-defective PSK (K57A) and PSK (745-1235), also produced nocodazole-resistant MTs, and PSK was found wherever MTs remained (Fig. 4, C-F). MT stabilization by PSK therefore requires protein localization but not catalytic activity, and this was again illustrated by the inability of PSK (1-940) to protect MTs from nocodazole (Fig. 4, G and H).
Tubulin undergoes several post-translational modifications, including the acetylation of ␣-tubulin on lysine residue 40; although this modification is a consequence, not a cause, of stabilization, the acetylation of ␣-tubulin is frequently used to distinguish between stable and dynamic forms of MTs (reviewed in Ref. 20). We therefore examined the effects of PSK on the levels of acetylated ␣-tubulin using anti-acetylated ␣-tubulin antibodies. The three forms of PSK that localize to MTs (PSK, kinase-defective PSK (K57A), and PSK (745-1235)) each increased levels of acetylated ␣-tubulin above those in neighboring uninjected cells (Fig. 5, A-F). The N-terminal kinase domain of PSK (1-940), which was unable to associate with MTs or generate nocodazole-resistant MTs, failed to stimulate the acetylation of tubulin (Fig. 5, G and H). Serum starvation of Swiss 3T3 cells seemed to result in a more cytoplasmic localization for full-length PSK, whereas C-terminal PSK (745-1235) remained tightly associated with the MTs under identical conditions (Fig. 5, A and E). Nocodazole-resistant and acetylated MTs are increased in Swiss 3T3 fibroblasts after serum stimulation (21,22); however, the PSK effects observed here occured in the absence of serum.
PSK Binds Tubulin in Vitro-Comparison of the C-terminal regulatory domain of PSK with other sequences present in the GenBank data base has shown that PSK does not share significant homology with any other proteins or with the MT-binding domains reported for MAPs, which contain a proline-rich sequence and three pseudorepeats (reviewed in Ref. 17). The ability of the C terminus of PSK (745-1235) to localize to MTs and produce stabilized perinuclear MT cables led us to investigate whether these sequences were able to interact with ␣or ␤-tubulin in vitro. A C-terminal fragment of PSK fused to glutathione-S-transferase (GST) was prepared, and recombinant PSK (amino acids 1064 -1235) was able to bind ␣and ␤tubulin in vitro, suggesting that PSK may interact directly with MTs (Fig. 6, lanes 1-3).
Taxol Dissociates PSK from MTs-The ability of PSK to bind and stabilize MTs led us to examine the effect of Taxolstabilized MTs on PSK. Swiss 3T3 cells expressing PSK were incubated in the presence of Taxol for 30 min, and cultures were fixed and costained for PSK and MTs. Fig. 7 shows that the addition of Taxol causes PSK to dissociate from MTs and become cytoplasmic (Fig. 7, A and B), whereas the MTs formed a separate ring around the nucleus (Fig. 7, C and D). These results demonstrate that PSK can respond to Taxolinduced changes in MT dynamics and that PSK might dissociate from MTs that are sufficiently stable.
PSK Phosphorylates Tubulin-The localization of PSK to MTs brings the kinase into the local vicinity of a number of potential MT-associated substrates that are regulated by phosphorylation, and these proteins could lead to additional affects of PSK on MTs. Because PSK binds tubulin, we investigated the ability of PSK to phosphorylate tubulin. Plasmids encoding PSK or PSK (K57A) were transiently transfected into cell cultures, and immune complexes of each protein were assayed for in vitro kinase activity using ␣and ␤-tubulin as substrates. The isoforms of tubulin were separated from each other using SDS-PAGE/6 M urea, and we found that PSK, but not kinasedefective PSK (K57A), phosphorylated ␣and ␤-tubulin (Fig.  8A, lanes 2 and 3).
Interestingly, addition of the MT-stabilizing agent Taxol rapidly down-regulated the ability of PSK and, to a lesser extent PSK (1-940), to phosphorylate tubulin, demonstrating that the catalytic activity of PSK responds to Taxol-induced changes in MT stability (Fig. 8B, lanes 2, 3, 5, and 6). In addition, transfected PSK, which is normally expressed in cells as a protein doublet of 185 and 165 kDa (3), was converted to the smaller form of the protein in the presence of Taxol (Fig. 8B, lanes 2 and  5). Because 9E10 antibody detects the N-terminal MYC-epitope tag on both forms of PSK (3), it is likely that the 185-kDa form of the protein is either truncated at the C terminus to generate a protein of 165 kDa that potentially lacks MT localization sequences; alternatively, PSK might undergo a change in posttranslational modification. In contrast, nocodazole had no effect on either the kinase activity or mobility of PSK (data not shown). DISCUSSION We have shown that the STE20-like kinase PSK localizes to MTs and produces stabilized perinuclear MT cables that are nocodazole-resistant and contain increased levels of acetylated ␣-tubulin. The N-terminal kinase domain of PSK (1-940) is unable to bind MTs or regulate their organization or stability, demonstrating that the association between PSK and MTs is required for these MT alterations to occur. The C terminus of PSK (745-1235) contains the MT localization and regulatory domain and recombinant PSK (1064 -1235) binds ␣and ␤-tubulin in vitro. The catalytic activity of PSK is not required for these effects on MTs because kinase-defective PSK (K57A) associates with MTs and regulates their organization and stability. The MT-stabilizing drug Taxol dissociates PSK from MTs and down-regulates the protein's catalytic activity, suggesting that PSK is sensitive to Taxol-induced changes in MT stability.
The stabilized perinuclear MT cables generated by PSK are similar to the MT structures produced by another STE20, X-PAK5, which binds MTs and induces stabilized curly MTs that form whorls around the nucleus (11). X-PAK5, like PSK, binds to MTs via its non-catalytic regulatory domain, and neither the stabilization nor the reorganization of MTs require the protein's catalytic activity (11). The Rho effector Rho kinase ␣ and DCAMKL1 also generate MT clusters around the nucleus independently of their kinase activity, and another non-enzymatic protein, mDia, co-localizes and stabilizes MTs orientated toward the wound edge (23,24). Interestingly, the ability of X-PAK5 to bind MTs is negatively regulated by its kinase activity because constitutively activated X-PAK5 is unable to bind MTs and relocates to the cytoplasm, where it causes dissolution of actin stress fibers and cell retraction (11). PSK also down-regulates actin stress fibers and induces cell rounding. and another STE20 SLK associates with MTs located at adhesion sites and reduces actin stress fibers (3,16). Interestingly, the catalytic activity of PSK is required for its effects on actin. and it is plausible that the kinase activity of PSK might act in a manner similar to X-PAK5 and regulate its localization and function (3). How PSK kinase activity is regulated in vivo is not known, but we have been unable to detect clear differences between the localization of PSK and kinase-defective PSK (K57A) in injected cells. PSK does, however, become more cytoplasmic when cells are starved of serum, whereas the C terminus of PSK (745-1235) lacking the kinase domain, remains tightly bound to MTs. The identification of upstream regulators for PSK and its kinase activity will be required to explore these possibilities further.
Catalytic activity is not required by PSK to re-organize and stabilize MTs, but the localization of PSK to MTs would be expected to bring the kinase into the vicinity of a number of potential MT-associated substrates. PSK could therefore have additional regulatory affects on MTs that require its catalytic activity. PSK phosphorylates ␣and ␤-tubulin, and others have shown that Syk and Src phosphorylate tubulin (25,26). The physiological significance of this modification and the sites of phosphorylation are unknown, but it has been suggested that the phosphorylation of tubulin could alter its binding properties to signaling proteins via SH2 domain-mediated interactions and/or regulate the accessibility of kinases to their substrates (25,26).
Although a number of kinases can regulate the activity and function of MT components, such as MAPs, by phosphorylation, only a few kinases have been shown to localize to MTs. Interestingly, MT-associated kinases include several components of MAPK signaling pathways, such as JNK and ERK (MAPKs), MAP kinase/ERK kinase (MEK), mixed-lineage kinase, X-PAK5 and SLK (MAPK kinase kinase kinases) (11,16,(27)(28)(29). PSK can activate JNK (3), as can its rat homolog, Tao2 (30), and it is therefore possible that this occurs on MTs. Whether JNK is involved in regulating MT stability remains to FIG. 8. PSK phosphorylates ␣and ␤-tubulin and is down-regulated by Taxol. A, growing COS-1 cells were transfected with either PSK (lanes 1 and 2), kinase-defective PSK (K57A) (lane 3), or pRK5 vector (lane 4). After 36 h, cell lysates were immunoprecipitated with anti-PSK antibodies, and immune complexes were subjected to an in vitro kinase assay using ␣and ␤-tubulin as substrate. Tubulin was omitted from lane 1 as a control. Tubulin subunits were separated by SDS-PAGE/6 M urea, and the subunits were identified by immunoblotting with ␣or ␤-tubulin specific antibodies (DM1A or TUB2.1, respectively). B, human embryonic kidney 293 cells were transfected with either pRK5-MYC vector alone (lanes 1 and 4), pRK5-MYC-PSK (lanes 2 and 5), or pRK5-MYC-PSK (1-940) (lanes 3 and 6). After 36 h, cells were treated with (lanes 4, 5, and 6) or without (lanes 1, 2, and 3) 10 M Taxol for 30 min. PSK was immunoprecipitated using anti-PSK antibodies, and immune complexes were subjected to an in vitro kinase assay using bovine ␣and ␤-tubulin as substrate. Immunoprecipitated PSK was detected by Western blotting. be determined, but JNK, ERK, p38, and glycogen synthase kinase-3␤ can each phosphorylate MAPs, such as tau, potentially regulating its affinity for MTs and their stability (31). Moreover, the MT-stabilizing agent Taxol activates JNK via mixed-lineage kinases such as MAP kinase/ERK kinase 1 and apoptosis-regulating kinase (32,33), but our finding that Taxol down-regulates the activity, expression, and localization of PSK indicates that PSK is unlikely to contribute to Taxolinduced JNK activation. Interestingly, ERK kinase activity has been shown to decrease the stability of MTs (34). In contrast, phosphorylation and inactivation of the MT-destabilizing protein stathmin stabilizes MTs, and the STE20 PAK can modulate the phosphorylation of stathmin (19); however, we have found that immune complexes of PSK were unable to phosphorylate stathmin on Ser-16, -25, or -38 (data not shown), suggesting that PSK does not regulate the stability of MTs via stathmin. Another MT-associated protein, disheveled, generates stabilized and acetylated MTs by inhibiting glycogen synthase kinase-3␤ and reducing its ability to phosphorylate MAPs (35). Dishevelled activates JNK (36), as does PSK (2), but the effects of PSK on glycogen synthase kinase-3␤ are unknown. PSK does bind directly to MTs in vitro, whereas glycogen synthase kinase-3␤ has been shown to bind MTs indirectly via its association with the neuronal MAP tau, and JNK may interact with MTs via JNK-interacting proteins (37,38).
The ability of PSK, SLK, and X-PAK5 to regulate MTs and actin filaments suggests that these STE20 proteins could function in the regulation of both cytoskeletal networks. Similarly, there is growing evidence that Rho family members (Rho, Rac, and Cdc42) may coordinately regulate both networks (39). MT growth activates Rac, itself a MT binding protein, and promotes actin polymerization and lamellipodial protrusion, whereas MT disassembly stimulates Rho and the formation of actin stress fibers and focal adhesions (40,41). The Rho guanine nucleotide exchange factors (GEFs), p190 Rho GEF, GEF-H1, and Lfc bind MTs and may locally activate Rho (42)(43)(44). Lfc activates JNK via STE20s such as PAK or mixed-lineage kinase (43), and it is possible that PSK, which activates JNK, might also interact with Lfc or another MT-associated Rho GEF. In addition, X-PAK5, which contains a CRIB domain, is targeted away from MTs toward actin-rich structures by Rac and Cdc42, and PSK might be regulated in a similar manner by small GTP-binding proteins that do not require a CRIB domain (11).
In conclusion, MTs play important roles in the regulation of cell morphogenesis, division, migration, and vesicle trafficking, and the ability of MTs to undergo changes in their stability and dynamics are crucial for their function. Here we show for the first time that a member of the GCK family of protein kinases, which lack CRIB domains, can localize to MTs and regulate their stability and organization. These findings imply a functional role for PSK, and perhaps other GCKs, in the regulation of MT dynamics.