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Departments of Molecular and Cellular Oncology,1 Molecular Genetics,2 Pathology, University of Texas M. D. Anderson Cancer Center, Houston, Texas,4 Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana3
Received 18 October 2004/ Returned for modification 26 November 2004/ Accepted 27 January 2005
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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/ß-tubulin heterodimers) as an interacting substrate of Pak1. Pak1 directly phosphorylated TCoB in vitro and in vivo on serines 65 and 128 and colocalized with TCoB on newly polymerized microtubules and on centrosomes. TCoB interacted with the GTPase-binding domain of Pak1 and activated Pak1 in vitro and in vivo. In contrast to wild-type TCoB, an S65A, S128A double mutant and knock-down of the endogenous TCoB or Pak1 reduced microtubule polymerization, suggesting that Pak1 phosphorylation is necessary for normal TCoB function. Overexpression of TCoB dramatically increased the number of 
-tubulin-containing microtubule-organizing centers, a phenotype reminiscent of cells overexpressing Pak1. TCoB was overexpressed and phosphorylated in breast tumors. These findings reveal a novel role for TCoB and Pak1 in regulating microtubule dynamics. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Microtubule subunits are heterodimers composed of one -tubulin polypeptide and one ß-tubulin polypeptide (7). Correct incorporation of these subunits into the polymer requires a complex folding process and is facilitated by a subfamily of chaperones known as CCT/TriC/c-cpn and a set of tubulin-specific cofactors (A to E) (11, 24). CCT-discharged quasi-native -tubulin and ß-tubulin are captured and stabilized by cofactors B and E or cofactors A and D, respectively (25). Cofactor-tubulin complexes associate to form a supercomplex containing tubulin cofactor C (TCoC), TCoD, TCoE, and - and ß-tubulin; GTP hydrolysis by the bound tubulin triggers the release of native /ß heterodimers (23). TCoC, TCoD, and TCoE all function in the assembly of the tubulin heterodimers, while TCoA and TCoB are specific to ß- and -tubulin, respectively (21).
To identify novel Pak1-interacting proteins with potential functions in the microtubule-mitotic axis, we performed a yeast two-hybrid screen of a human mammary gland cDNA expression library. We have identified TCoB (GenBank accession number XM_009339) as a novel Pak1-interacting substrate. Using various biochemical, molecular, and cell biology approaches, we have provided evidence that Pak1 phosphorylation of TCoB is essential for the polymerization of new microtubules and thus provides novel insights into the molecular mechanism of Pak1 regulation of microtubules.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Plasmid construction. Red fluorescent protein (RFP)-, glutathione S-transferase (GST)- and T7-tagged TCoB constructs were generated by amplifying the selective region of TCoB from the two-hybrid clones obtained from the library screening and by subcloning this region into DsRed2 (Clontech), pGEX 5X (Amersham), and pcDNA3.1 (Invitrogen) vectors, respectively. TCoB-S65A, TCoB-S128Ala, and TCoB-S65AS128A double mutations were generated by site-directed mutagenesis by using a quick-change mutagenesis kit from Stratagene. By amplifying the indicated regions via PCR, we created TCoB fragments and subcloned them into pGEX vectors.
Cell cultures, reagents, transfection, and cell extracts.
MCF-7, SKBR3, and MCF-7/HER2 human breast cancer cells and IMR90 human lung fibroblasts were maintained in Dulbecco's modified Eagle's medium-F12 (1:1) supplemented with 10% fetal calf serum. Antibodies against Pak1 were purchased from Zymed Laboratories Inc. and Cell Signaling; antibodies against T7 were purchased from Novagen; antibody against -tubulin and Glu was obtained from Sigma; antibody for Pak2 and Pak3 was purchased from Santa Cruz; antibody against phosphorylated (phospho-T423) Pak1 was described previously (10). Transient transfection studies were performed with a FuGENE 6 kit (Roche Biochemical) in accordance with the manufacturer's instructions. Small interfering RNA (siRNA) against TCoB was designed and synthesized by QiagenXeragon against TCoB target sequence 5'-AATGGGAAACGCTACTTCGAA-3'. siRNA transfections were performed by using Oligofectamine (Invitrogen) according to the manufacturer's protocol. siRNA against Pak1 (catalog number 6361), siRNA against Pak2 (catalog number 6366), and control fluorescein isothiocyanate siRNA (catalog number 6201) were purchased from Cell Signaling. Phosphoserine antibody (catalog number 37430) was purchased from QIAGEN. Anti-peptide antibody (rabbit) against TCoB was generated against the C-terminal 14 amino acids (aa 231 to 244 [NH3-VGDFPEEDYGLDEI-COOH]) and was affinity purified by using GST-TCoB.
Immunoblotting. For preparation of cell extracts, cells were washed three times with phosphate-buffered saline (PBS) and lysed in buffer (50 mM Tris-HCl [pH 7.5], 120 mM NaCl, 1% Triton X-100, 100 mM NaF, 200 mM NaVO5, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail [GIBCO]) for 15 min on ice. The lysates were centrifuged with an Eppendorf centrifuge at 4°C for 15 min. Cell lysates containing equal amounts of protein were then resolved on sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred onto nitrocellulose, and probed with the appropriate antibodies using an enhanced chemiluminescence method. Cell lysates for immunoprecipitation were prepared by using Nonidet P-40 (NP-40) lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 0.5% NP-40, 1x protease inhibitor mixture, 1 mM sodium vanadate). Immunoprecipitation was performed for 2 h at 4°C using 1 µg of antibody/mg of protein.
Pak1 kinase assay.
Using myelin basic protein (MBP) or GST-TCoB fusion protein, we performed in vitro kinase assays in HEPES buffer (50 mM HEPES, 10 mM MgCl2, 2 mM MnCl2, 1 mM dithiothreitol) containing 100 ng of purified GST-Pak1 enzyme, 10 µCi of [-32P]ATP, and 25 µM cold ATP. MBP was purchased from Upstate Biotechnology. GST proteins were purified by using glutathione Sepharose (Amersham Biosciences) according to the manufacturer's instructions. The reaction was carried out in a volume of 30 µl for 30 min at 30°C and then stopped by the addition of 10 µl of 4x SDS buffer. We analyzed the reaction products by SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiographed them. In some in vitro kinase assays, MBP (20 µg) was added to the reaction. For stimulation of Pak1 autophosphorylation, purified GST-Cdc42 (6.7 mM) was preloaded with 0.18 mM GTP-S for 10 min in a volume of 20 µl in 20 mM Tris buffer (pH 7.5) containing 50 mM NaCl. The activated Cdc42 or purified GST-TCoB (6.7, 13.3, or 20 mM) was then incubated with Pak1 enzyme in HEPES buffer containing 1 µl of ATP in a final reaction volume of 30 µl. Purified human Pak1 enzyme (catalog number GLO110-100) and Cdc42 (catalog number GLO128-001) were purchased from Alexis Biochemicals. His-BAD (catalog number 14-357) was purchased from Upstate biotechnology and used as a positive control.
GST pull-down assay. In vitro transcription and translation of the Pak1 and TCoB proteins were performed by using the TNT-coupled transcription-translation system (Promega) as described previously (10). In brief, using a T7-TNT kit (Promega), we translated 1 µg of cDNA in the pcDNA 3.1 vector in vitro in the presence of [35S]methionine in a reaction volume of 50 µl. The reaction mixture was diluted to 1 ml with NP-40 lysis buffer (25 mM Tris, 50 mM NaCl, 1% NP-40). An aliquot (250 µl) was used for each GST pull-down assay. Translation and product size were verified by subjecting 2 µl of the reaction mixture to SDS-PAGE and autoradiography. The GST pull-down assays were performed by incubating equal amounts of GST, GST-Pak1, and GST-TCoB fusion proteins immobilized on GST Sepharose beads (Amersham Biosciences) with in vitro-translated 35S-labeled Pak1 or TCoB protein. Bound proteins were isolated by incubating the mixture for 2 h at 4°C, washing five times with NP-40 lysis buffer, eluting the proteins with 2x SDS buffer, and separating the proteins by SDS-PAGE. The bound proteins were then visualized by fluorography.
Immunofluorescence and confocal microscopy studies. We determined the cellular localization of proteins by indirect immunofluorescence as described previously (10). Briefly, cells grown on glass coverslips were fixed in methanol at –20°C for 4 min. The cells were incubated with primary antibodies for 2 h, washed three times in PBS, and then incubated with secondary antibodies conjugated with Alexa 546 (red), Alexa 633 (blue), or Alexa 488 (green) dye from Molecular Probes (Eugene, Oreg.). The DNA dye Topro-3 (Molecular Probes) was used to costain the DNA (blue). Cells treated only with the secondary antibodies or when available with the peptide against which the primary antibody was raised served as controls.
Confocal scanning analysis was performed by using either a Zeiss LSM510 or an Olympus FV300 laser scanning confocal microscope in accordance with established methods, utilizing sequential laser excitation to minimize the possibility of fluorescent emission bleed-through. Each image is a three-dimensional reconstructed image of stacks of serial Z sections at the same cellular level and magnification. Colocalization of two proteins is shown as yellow for red and green, turquoise for green and blue, pink for red and blue, and white for the overlap of three-color fluorescence. Images were exported to Microsoft Photo Editor for adjustment of contrast prior to assembly into figures using Microsoft Power Point.
Nocodazole treatments for microtubule biogenesis assays were performed as previously described (4, 28). Briefly, cells were treated with 4 µM nocodazole for 30 min, rinsed twice with PBS, and then incubated in medium with 10% serum for 15 min to allow for new microtubule biogenesis. Cells were then washed in PHEM cytoskeletal buffer {100 mM PIPES [piperazine-N,N'-bis(2-ethanesulfonic acid)], 100 mM HEPES, 1 mM EGTA, 2 mM MgSO4, 0.2% Triton X-100 (pH 7.0)} for 2 min to extract unpolymerized tubulin and then processed as described above. A mouse monoclonal antibody against tyrosinated microtubules (selectively present on newly polymerized microtubules) was used for colocalization of TCoB or Pak1 selectively with this microtubule population. In siRNA experiments, attenuated microtubule regrowth was defined as siRNA-positive cells with less than 25% abundance of newly formed, tyrosinated microtubules compared with neighboring, untransfected cells.
Immunohistochemistry. For immunohistochemical detection of Pak1 and TCoB, sections were deparaffinized with xylene and rehydrated with graded ethanol. Sections were incubated in 0.3% H2O2 and methanol for 30 min to inactivate endogenous peroxidase. The sections were then boiled for 10 min in 0.01 M citrate buffer and cooled for 30 min at room temperature to expose antigenic epitopes. The sections were incubated with 2% normal goat serum in 1% bovine serum albumin and PBS for 30 min and then incubated with anti-Pak1 (Cell Signaling) at a 1:25 dilution and peptide affinity-purified anti-TCoB at a 1:10 dilution overnight and at room temperature. The sections were washed three times with 0.05% Tween in PBS for 10 min and incubated with secondary antibody and then developed with diaminobenzidine-H2O2 and counterstained with Mayer's hematoxylin.
Reproducibility. The results presented are representative of at least three replicate experiments.
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RESULTS AND DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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We next examined the localization of endogenous TCoB by using the affinity-purified TCoB antibody. A significant amount of endogenous TCoB was observed to colocalize with microtubules (Fig. 1F). However, exclusive localization of TCoB at the plus ends of microtubules was not observed, indicating that the TCoB cytoskeleton-associated protein, glycine-rich (CAP-GLY) motif is not sufficient to confer the plus-end tracking activity observed with other CAP-GLY-containing proteins and that TCoB may have another microtubule-associated function outside of heterodimer assembly.
TCoB is a Pak1 physiological substrate. Because Pak1 is a serine/threonine kinase, we examined whether TCoB is a substrate of Pak1. MCF-7 cells were metabolically labeled with [32P]orthophosphoric acid, and the status of TCoB phosphorylation was determined after treatment with physiological signals that activate Pak1, including epidermal growth factor (EGF), sphingosine, and serum (2). Results show that signals that activate Pak1 increase the phosphorylation of both endogenous TCoB (Fig. 2A) and epitope-tagged T7-TCoB (Fig. 2B).
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To demonstrate the functional significance of Pak1 in the phosphorylation of TCoB during microtubule biogenesis, we selectively knocked down Pak1 with a Pak1-specific siRNA. Down-regulation of Pak1 expression substantially reduced the phosphorylation of TCoB in the nocodazole regrowth assay (Fig. 2E, compare lane 2 to lane 3). To examine whether TCoB is a direct substrate of Pak1, we performed an in vitro kinase assay using purified GST-TCoB and purified Pak1 enzyme (Fig. 2F). The protein BAD, an established Pak1 substrate (18), was used as a positive control. The results showed that the Pak1 enzyme could phosphorylate TCoB and BAD in vitro in a similar fashion. The autophosphorylation of Pak1 enzyme was substantially higher when GST-TCoB was used as a substrate than when GST alone or His-BAD was used (Fig. 2F, middle panel, compare lanes 1 and 3 with lane 2).
We next mapped the Pak1 phosphorylation sites in TCoB by using GST fusions of various TCoB domains. Deletion of TCoB N-terminal aa 1 to 33 (Fig. 2G, lane 3) or the CAP-GLY domain (aa 140 to 244) (Fig. 2G, lane 6) had no effect on the ability of Pak1 to phosphorylate TCoB. However, deletion of the N-terminal aa 1 to 89, which contain the N-terminal functional region, partially reduced the ability of Pak1 to phosphorylate TCoB (50% reduction) (Fig. 2G, lane 4). Deletion of aa 1 to 139, which contain the N-terminal functional region and part of the coiled-coil region, severely reduced the ability of Pak1 to phosphorylate TCoB (Fig. 2G, lane 5). The reduced ability of Pak1 to phosphorylate a TCoB mutant lacking aa 1 to 89 and the complete loss of Pak1 phosphorylation in a TCoB mutant lacking aa 1 to 139 suggested that Pak1 phosphorylated TCoB at at least two or more sites.
Examination of TCoB aa 1 to 140 revealed two potential Pak1 consensus phosphorylation sites, serine 65 and serine 128. Pak1 phosphorylates a number of substrates on serine/threonine residues, preferably in the context of preceding basic residues such as (K/R)(R/X)X(S/T) (2, 9). Single-point mutation of either serine to alanine partially reduced the ability of Pak1 to phosphorylate TCoB (Fig. 2H, lanes 2 and 3, respectively). Double mutation of both serines to alanine in the full-length TCoB severely reduced the phosphorylation in vitro (Fig. 2H, lane 4) and nearly abolished TCoB phosphorylation in vivo upon stimulation with EGF (Fig. 2I).
TCoB and Pak1 interaction domains.
To identify the domains of the Pak1-TCoB interaction, we examined the ability of in vitro-translated Pak1 to bind with the full-length TCoB as well as various TCoB domains expressed as GST fusion proteins. An earlier study identified three functional domains in TCoB: the N-terminal functional domain (aa 1 to 118); the central coiled-coil domain (aa 119 to 163), which is required for maintenance of cellular -tubulin levels; and the C-terminal CAP-GLY domain (aa 164 to 244), which is required for binding of -tubulin (16). Our results showed that wild-type TCoB interacted with Pak1 (Fig. 3A, lane 2). Deletion of parts of the N-terminal functional domain had no effect on the binding of TCoB to Pak1 (Fig. 3A, lanes 3 and 4). However, partial deletion of the central coiled-coil region abolished TCoB binding with Pak1 (Fig. 3A, lane 5). The C-terminal CAP-GLY domain alone did not show any interaction with Pak1 (Fig. 3A, lane 6). These results suggested that the Pak1-binding site in TCoB was localized in the central coiled-coil domain.
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Because the TCoB-interacting surface of Pak1 overlaps with the CRIB domain and because GTPase binding to the CRIB domain has been shown to activate Pak1 by relieving the autoinhibition of Pak1 (2), we examined whether the interaction between Pak1 and TCoB would relieve Pak1 autoinhibition and thereby increase its activity. We first compared the TCoB-mediated activation of Pak1 with a known activator of Pak1 Cdc42. Results showed that incubation of both Cdc42 and TCoB with the Pak1 enzyme activated Pak1 autophosphorylation (Fig. 3C). However, TCoB activation of Pak1 was less than that of Cdc42 in this in vitro assay, as substantially more TCoB protein was required to achieve a level of activation similar to that of Cdc42 (Fig. 3C). To examine whether the TCoB-mediated increase in autophosphorylation translates into increased Pak1 activity to exogenous substrates, we incubated GST or GST-TCoB with Pak1 enzyme and used MBP as a substrate to measure Pak1 activity. Interestingly, TCoB increased Pak1 activity against the established substrate MBP in a dose-dependent manner (Fig. 3D). Furthermore, as we expected from the data illustrated in Fig. 3C, Pak1 autophosphorylation was markedly increased (Fig. 3D, left panel). Pak1 binding to TCoB was essential, as deletion of the Pak1-binding domain in TCoB (aa 1 to 139) abolished the ability of TCoB to activate Pak1 (Fig. 3E). By exploring whether TCoB stimulates Pak1 kinase activity in vivo, we demonstrated that transient coexpression of T7-TCoB, but not a mutant TCoB lacking the Pak1-binding domain (aa 1 to 139), enhanced Pak1 kinase activity (Fig. 3F).
TCoB phosphorylation mutant blocks polymerization of new microtubules.
We next reasoned that if the TCoB-Pak1 interaction is playing a role in microtubule assembly, there should be increased association during periods of rapid microtubule assembly, such as during cell recovery from treatment with nocodazole, an established microtubule-disrupting agent. Indeed, TCoB and Pak1 were coimmunoprecipitated during this recovery phase (Fig. 4A). We then examined the association of TCoB with newly polymerized microtubules by using confocal microscopy and an established microtubule regrowth assay (4, 28) (Fig. 4B). Since newly polymerized microtubules contain tyrosinated -tubulin, we used an antibody specific for tyrosinated tubulin (blue) as a marker of these newly polymerized microtubules. Colocalization of TCoB (red) with tyrosinated tubulin (blue) was demonstrated by the appearance of a pink color. We found that nocodazole treatment completely depolymerized microtubules (data not shown). However, 15 min after the cells were washed and free of nocodazole, we observed extensive TCoB protein localization along the growing microtubules during recovery from nocodazole (Fig. 4B, arrows). Our results revealed that endogenous TCoB localizes to newly polymerized microtubules and colocalizes with the tyrosinated -tubulin microtubules.
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-tubulin (green) were localized by confocal microscopy. The majority of either transfected wild-type or mutant RFP-TCoB was localized along the tyrosinated tubules, while a portion seemed to converge in larger aggregates. This localization pattern was also observed for endogenous TCoB, with some cell-to-cell variation (data not shown). Microtubule regrowth was observed in wild-type RFP-TCoB-expressing cells (Fig. 4C), while 80% of the cells expressing the RFP-TCoB S65A, S128A double mutant (which lacks both Pak1 phosphorylation sites) showed attenuated microtubule regrowth compared to neighboring untransfected cells (Fig. 4D). To rule out any possible experimental artifacts resulting from the RFP tag, which can oligomerize when expressed using certain vectors that were not employed in these experiments, these experiments were repeated using wild-type and mutant T7-tagged TCoB (Fig. 4E and F). Similar to the RFP-tagged mutant TCoB, cells transfected with wild-type T7-TCoB appeared similar to neighboring untransfected cells (Fig. 4E), while cells transfected with the T7-tagged TCoB phosphorylation mutant showed attenuated synthesis of new microtubules (Fig. 4F). These results suggested that phosphorylation of TCoB at both sites was important for the regrowth of new microtubules.
Role of Pak1-TCoB interaction in polymerization of new microtubules.
To assess the significance of the endogenous TCoB and Pak1 interactions in the maintenance of cellular -tubulin levels and polymerization of new microtubules, we have used a knock-down approach using siRNA specific to Pak1 and TCoB. TCoB-specific siRNA targeted to two different regions of the TCoB mRNA reduced the expression of endogenous TCoB mRNA (Fig. 5A) and protein (Fig. 5B) by greater than 80%, with no or minimal alterations in the -tubulin protein levels in the cells (Fig. 5B, left panel). Similar results were obtained using siRNA targeting to a different site of TCoB (Fig. 5B, right panel). Also, knock down of Pak1 using two distinct Pak1-specific siRNAs efficiently reduced Pak1 expression but did not affect cellular -tubulin levels (Fig. 5C). These results suggest that Pak1 and TCoB interactions are not essential for the maintenance of -tubulin levels in human cells or that the minimal levels of TCoB present in the cells following siRNA may be enough to maintain the cellular TCoB levels. Previously published experiments with yeast also showed that TCoB knock down did not severely affect the cellular -tubulin levels. Although tubulin-folding cofactor mutants in yeast caused an altered microtubule cytoskeleton, the defects are not clearly attributable to decreases in the steady-state levels of - or ß-tubulin (16). Also, given that in fission yeast, TCoE was shown to bypass the requirement of TCoB (15), it is possible that TCoE may perform a similar role in mammalian cells and thus may account for the maintenance of -tubulin levels in TCoB siRNA-treated cells.
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We next examined the effect of the TCoB and Pak1 knock down on the polymerization of new microtubules. In order to verify expression of TCoB and Pak1 in IMR90 cells and the effectiveness of specific siRNAs in these cells, we transfected either TCoB-specific or Pak1-specific siRNA and examined target protein expression by Western blot (Fig. 5E). Results show that endogenous TCoB and Pak1 expression in IMR90 cells was effectively reduced by transfecting specific siRNA.
Using IMR90 cells as a model system for microtubule changes, cells were transfected separately with Pak1 and TCoB siRNA to validate our biochemical results. Pak2 siRNA and nonspecific control siRNA were used as controls. Pak2 expression in IMR90 cells was validated by Western blot (data not shown). All transfected cells appeared to be viable, with no apparent morphological apoptotic changes either before or after nocodazole treatment (data not shown). Knock down of either TCoB or Pak1 severely reduced the formation of new microtubules (Fig. 5F) in the nocodazole-based microtubule biogenesis assay described above. More than 70% of cells (n = 100) transfected with siRNA against TCoB showed reduced regrowth of microtubules compared with untransfected neighboring cells 15 min after nocodazole removal (Fig. 5F, upper left panel). Similarly, Pak1 knock down also reduced microtubule regrowth in the transfected cells in the nocodazole treatment assay (Fig. 5F, upper right panel). Knock down with control, nonspecific siRNA (Fig. 5F, lower left panel) or knock down of Pak2 by Pak2-specific siRNA (Fig. 5F, lower right panel) did not show any apparent impact on the ability of endogenous TCoB to promote microtubule formation or on TCoB colocalization with newly formed microtubules. Thus, our findings indicated that both Pak1 and TCoB were critical molecules for the generation of new microtubules and that Pak1 phosphorylation of TCoB might be essential for normal function of TCoB.
Role of TCoB in mitotic spindle dynamics.
During colocalization studies with endogenous TCoB antibody, we observed colocalization between TCoB and tubulin during interphase and also during mitosis, where TCoB association with staining of the mitotic spindle was prominent (Fig. 6A). Because TCoB is a substrate of Pak1 and since TCoB overexpression increases Pak1 activity (Fig. 3C to F), we examined whether TCoB overexpression could mimic the Pak1 phenotypes (i.e., abnormalities in mitotic spindles) that we had previously shown to be the direct result of Pak1 overexpression (26). Human IMR90 cells were transfected with RFP-TCoB (red) and stained for -tubulin (green) as a marker of the centrosomes (Fig. 6B). Thirty percent of the RFP-TCoB-transfected cells exhibited more than two -tubulin-containing MTOCs (data not shown). We validated these findings in several mammary epithelial model cell lines (MCF-7, SKBR3, and MCF-7 overexpressing HER2). Overexpression of RFP-TCoB resulted in the appearance of 70% of the transfected SKBR3 cells with more than two MTOCs (Fig. 6C) and in 30% of transfected MCF-7 cells with more than two MTOCs (Fig. 6C). Because SKBR3 cells are known to naturally overexpress HER2, and HER2 signaling is known to activate Pak1 (9), we hypothesized that hyperactivation of Pak1 in SKBR3 cells may contribute to the TCoB-induced increase in the number of MTOCs. Indeed, transfection of RFP-TCoB into MCF-7 clones overexpressing HER2 increased the number of MTOCs to levels similar to those observed in SKBR3 cells (Fig. 6C). Transfection of the RFP-TCoB S65A, S128A mutant that cannot be phosphorylated by Pak1 did not induce multiple MTOCs (Fig. 6C). Furthermore, cotransfection of the Pak1 autoinhibitory fragment (Pak1 amino acids 83 to 149) or Pak1 siRNA along with RFP-TCoB abolished the ability of TCoB to induce multiple MTOCs in MCF-7 cells (Fig. 6C), suggesting that the presence of more than two -tubulin dots may be due in part to a failure in cytokinesis in addition to other mitotic events such as centrosome duplication.
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In conclusion, this report identifies for the first time TCoB as a Pak1-interacting substrate and provides new insight into how Pak1 and TCoB influence microtubule biogenesis. Our results suggest that Pak1 phosphorylates TCoB and that such phosphorylation has a role in the growth of new microtubules. Furthermore, these newly identified roles for TCoB and Pak1 in microtubule dynamics and normal cell function may be coordinately deregulated in human cancers.
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ACKNOWLEDGMENTS |
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We thank Liana Adam for the -tubulin confocal microscopy and Sally A. Lewis, New York University Medical Center, for providing cofactor B pet23d vector.
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FOOTNOTES |
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