A functional cooperativity between Aurora A kinase and LIM kinase1: Implication in the mitotic process

Aurora kinase A (Aur-A), a mitotic kinase, regulates initiation of mitosis through centrosome separation and proper assembly of bipolar spindles. LIM kinase 1 (LIMK1), a modulator of actin and microtubule dynamics, is involved in the mitotic process through inactivating phosphorylation of cofilin. Phosphorylated LIMK1 is recruited to the centrosomes during early prophase, where it colocalizes with γ-tubulin. Here, we report a novel functional cooperativity between Aur-A and LIMK1 through mutual phosphorylation. LIMK1 is recruited to the centrosomes during early prophase and then to the spindle poles, where it colocalizes with Aur-A. Aur-A physically associates with LIMK1 and activates it through phosphorylation, which is important for its centrosomal and spindle pole localization. Aur-A also acts as a substrate of LIMK1, and the function of LIMK1 is important for its specific localization and regulation of spindle morphology. Taken together, the novel molecular interaction between these two kinases and their regulatory roles on one other''s function may provide new insight on the role of Aur-A in manipulation of actin and microtubular structures during spindle formation.Key words: LIMK1, Aurora A, mitotic spindle, phosphorylation     

Aurora A kinase modulates actin cytoskeleton through phosphorylation of Cofilin: Implication in the mitotic process

Aurora A kinase regulates early mitotic events through phosphorylation and activation of a variety of proteins. Specifically, Aur-A is involved in centrosomal separation and formation of mitotic spindles in early prophase. The effect of Aur-A on mitotic spindles is mediated by the modulation of microtubule dynamics and association with microtubule binding proteins. In this study we show that Aur-A exerts its effects on spindle organization through the regulation of the actin cytoskeleton. Aurora A phosphorylates Cofilin at multiple sites including S³ resulting in the inactivation of its actin depolymerizing function. Aur-A interacts with Cofilin in early mitotic phases and regulates its phosphorylation status. Cofilin phosphorylation follows a dynamic pattern during the progression of prophase to metaphase. Inhibition of Aur-A activity induced a delay in the progression of prophase to metaphase. Aur-A inhibitor also disturbed the pattern of Cofilin phosphorylation, which correlated with the mitotic delay. Our results establish a novel function of Aur-A in the regulation of actin cytoskeleton reorganization, through Cofilin phosphorylation during early mitotic stages.     

Phosphorylated LIM Kinases Colocalize with Gamma-Tubulin in Centrosomes During Early Stages of Mitosis

LIM kinases (LIMK1 and LIMK2) are LIM domain containing serine/threonine kinases that modulate reorganization of actin cytoskeleton through inactivating phosphorylation of cofilin. The Rho family of small GTPases regulates the catalytic activity of LIMK1 and LIMK2 through activating phosphorylation by ROCK or by p21 kinase. Recent studies have suggested that LIMK1 could play a role in modulation of cellular growth by alteration of the cell cycle in breast and prostate tumor cells; however, the direct mitogenic effects of LIMK1 in these tumor cells is yet to be elucidated. Via immunofluorescence, in this study, we show that phosphorylated LIM kinases (pLIMK1/2) are colocalized with γ-tubulin in the centrosomes during the early mitotic phases of human breast and prostate cancer cells (MDA-MB-231 and DU145); apparent colocalization begins in the centrosomes in prophase. As shown by both bright field (MDA-MB-231) and fluorescent immunohistochemistry (MDA-MB-231 and DU145), pLIMK1/2 does not localize to centrosomes during interphase. By bright field immunohistochemistry, the largest area of the centrosome that is stained with pLIMK1/2 occurs at anaphase. In early telophase, reduced staining of pLIMK1/2 at the spindle poles and concomitant accumulation of pLIMK1/2 at the cleavage furrow begins to occur. In late telophase, loss of staining of pLIMK1/2 and of colocalization with γ-tubulin occurs at the poles and pLIMK1/2 became further concentrated at the junction between the two daughter cells. Co-immunoprecipitation studies indicated that γ-tubulin associates with phosphorylated LIMK1 and LIMK2 but not with dephosphorylated LIMK1 or LIMK2. The results suggest that activated LIMK1/2 may associate with γ-tubulin and play a role in mitotic spindle assembly.     

Different activity regulation and subcellular localization of LIMK1 and LIMK2 during cell cycle transition

LIM kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through phosphorylating and inactivating cofilin, an actin-depolymerizing factor of actin filaments. Here, we describe a detailed analysis of the cell-cycle-dependent activity of LIMK2, and a subcellular localization of LIMK1 and LIMK2. The activity of LIMK2, distinct from LIMK1, toward cofilin phosphorylation did not change in the normal cell division cycle. In contrast, LIMK2 was hyperphosphorylated and its activity was markedly increased when HeLa cells were synchronized at mitosis with nocodazole treatment. Immunofluorescence analysis showed that LIMK1 was localized at cell-cell adhesion sites in interphase and prophase, redistributed to the spindle poles during prometaphase to anaphase, and accumulated at the cleavage furrow in telophase. In contrast, LIMK2 was diffusely localized in the cytoplasm during interphase, redistributed to the mitotic spindle, and finally to the spindle midzone during anaphase to telophase. These findings suggest that LIMK2 is activated in response to microtubule disruption, and that LIMK1 and LIMK2 may play different roles in regulating for the mitotic spindle organization, chromosome segregation, and cytokinesis during the cell division cycle.     

LIM kinase-mediated cofilin phosphorylation during mitosis is required for precise spindle positioning

The interaction of astral microtubules with cortical actin networks is essential for the correct orientation of the mitotic spindle; however, little is known about how the cortical actin organization is regulated during mitosis. LIM kinase-1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. LIMK1 activity increases during mitosis. Here we show that mitotic LIMK1 activation is critical for accurate spindle orientation in mammalian cells. Knockdown of LIMK1 suppressed a mitosis-specific increase in cofilin phosphorylation and caused unusual cofilin localization in the cell cortex in metaphase, instability of cortical actin organization and astral microtubules, irregular rotation and misorientation of the spindle, and a delay in anaphase onset. Similar results were obtained by treating the cells with a LIMK1 in hibitor peptide or latrunculin A or by overexpressing a non-phosphorylatable cofilin(S3A) mutant. Furthermore, localization of LGN (a protein containing the repetitive Leu-Gly-Asn tripeptide motifs), an important regulator of spindle orientation, in the crescent-shaped cortical regions was perturbed in LIMK1 knockdown cells. Our results suggest that LIMK1-mediated cofilin phosphorylation is required for accurate spindle orientation by stabilizing cortical actin networks during mitosis.     

Nek9 is a Plk1-activated kinase that controls early centrosome separation through Nek6/7 and Eg5

The NIMA-family kinases Nek9/Nercc1, Nek6 and Nek7 form a signalling module required for mitotic spindle assembly. Nek9, the upstream kinase, is activated during prophase at centrosomes although the details of this have remained elusive. We now identify Plk1 as Nek9 direct activator and propose a two-step activation mechanism that involves Nek9 sequential phosphorylation by CDK1 and Plk1. Furthermore, we show that Plk1 controls prophase centrosome separation through the activation of Nek9 and ultimately the phosphorylation of the mitotic kinesin Eg5 at Ser1033, a Nek6/7 site that together with the CDK1 site Thr926 we establish contributes to the accumulation of Eg5 at centrosomes and is necessary for subsequent centrosome separation and timely mitosis. Our results provide a basis to understand signalling downstream of Plk1 and shed light on the role of Eg5, Plk1 and the NIMA-family kinases in the control of centrosome separation and normal mitotic progression.     

Plk1 Regulates Both ASAP Localization and Its Role in Spindle Pole Integrity

Bipolar spindle formation is essential for faithful chromosome segregation at mitosis. Because centrosomes define spindle poles, abnormal number and structural organization of centrosomes can lead to loss of spindle bipolarity and genetic integrity. ASAP (aster-associated protein or MAP9) is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. Its phosphorylation by Aurora A is required for spindle assembly and mitosis progression. Here, we show that ASAP is localized to the spindle poles by Polo-like kinase 1 (Plk1) (a mitotic kinase that plays an essential role in centrosome regulation and mitotic spindle assembly) through the γ-TuRC-dependent pathway. We also demonstrate that ASAP is a novel substrate of Plk1 phosphorylation and have identified serine 289 as the major phosphorylation site by Plk1 in vivo. ASAP phosphorylated on serine 289 is localized to centrosomes during mitosis, but this phosphorylation is not required for its Plk1-dependent localization at the spindle poles. We show that phosphorylated ASAP on serine 289 contributes to spindle pole stability in a microtubule-dependent manner. These data reveal a novel function of ASAP in centrosome integrity. Our results highlight dual ASAP regulation by Plk1 and further confirm the importance of ASAP for spindle pole organization, bipolar spindle assembly, and mitosis.     

Phosphorylation of CHO1 by Lats1/2 regulates the centrosomal activation of LIMK1 during cytokinesis

Large tumor suppressor 1 and 2 (Lats1/2) regulate centrosomal integrity, chromosome segregation and cytokinesis. As components of the centralspindlin complex, the kinesin-like protein CHO1 and its splicing variant MKLP1 colocalize with chromosome passenger proteins and GTPases and regulate the formation of the contractile ring and cytokinesis; however, the regulatory mechanisms of CHO1/MKLP1 remain elusive. Here, we show that Lats1/2 phosphorylate Ser716 in the F-actin-interacting region of CHO1, which is absent in MKLP1. Phosphorylated CHO1 localized to the centrosomes and midbody, and the actin polymerization factor LIM-kinase 1 (LIMK1) was identified as its binding partner. Overexpression of constitutively phosphorylated and non-phosphorylated CHO1 altered the mitotic localization and activation of LIMK1 at the centrosomes in HeLa cells, leading to the inhibition of cytokinesis through excessive phosphorylation of Cofilin and mislocalization of Ect2. These results suggest that Lats1/2 stringently control cytokinesis by regulating CHO1 phosphorylation and the mitotic activation of LIMK1 on centrosomes.     

p53 localizes to the centrosomes and spindles of mitotic cells in the embryonic chick epiblast, human cell lines, and a human primary culture: An immunofluorescence study

Immunofluorescent staining of mitotic centrosomes and spindles by anti-p53 antibodies was observed in the embryonic chick epiblast by epifluorescence microscopy and in three human cancer cell lines, an SV40-immortalized cell line, and a normal human fibroblast culture by confocal microscopy. In the chick epiblast, the centrosomes stained from early prophase through to the formation of the G1 nuclei and the spindle fibers stained from prophase through to telophase. In the human cells, the staining was observed from late prophase to telophase. The epiblast was stained by the anti-p53 antibodies DO-1, Ab-6, and Bp53-12. The human cells were also stained by these antibodies as well as by other anti-p53 antibodies. Preabsorption of DO-1 and Bp53-12 with purified tubulin did not diminish the immunostaining, showing that the antibodies were not reacting with tubulin in the mitotic centrosomes and spindles. The immunostaining in the chick epiblast was very clearly localized to the mitotic centrosomes and spindles, revealing a cytoplasmic location for p53 during mitosis and accounting for earlier reports of an association between p53, tubulin, and centrosomes. The localization of p53 to the spindle supports an involvement of p53 in spindle function.     

Phosphorylation of centrin during the cell cycle and its role in centriole separation preceding centrosome duplication

Once during each cell cycle, mitotic spindle poles arise by separation of newly duplicated centrosomes. We report here the involvement of phosphorylation of the centrosomal protein centrin in this process. We show that centrin is phosphorylated at serine residue 170 during the G(2)/M phase of the cell cycle. Indirect immunofluorescence staining of HeLa cells using a phosphocentrin-specific antibody reveals intense labeling of mitotic spindle poles during prophase and metaphase of the cell division cycle, with diminished staining of anaphase and no staining of telophase and interphase centrosomes. Cultured cells undergo a dramatic increase in centrin phosphorylation following the experimental elevation of PKA activity, suggesting that this kinase can phosphorylate centrin in vivo. Surprisingly, elevated PKA activity also resulted intense phosphocentrin antibody labeling of interphase centrosomes and in the concurrent movement of individual centrioles apart from one another. Taken together, these results suggest that centrin phosphorylation signals the separation of centrosomes at prophase and implicates centrin phosphorylation in centriole separation that normally precedes centrosome duplication.     

The Xenopus laevis centrosome aurora/Ipl1-related kinase.

The cDNA encoding the protein kinase pEg2 was originally cloned through a differential screening performed during the early development of Xenopus laevis. pEg2 orthologues were found in various organisms and were classified in a new family of oncogenic mitotic protein kinases named 'aurora/Ipl1-related kinases' after the Drosophila melanogaster gene aurora and the Saccharomyces cerevisiae gene Ipl1. The catalytic activity of pEg2 is necessary for the mitotic microtubule spindle formation in Xenopus laevis egg extracts. The addition of a dominant negative form of pEg2 to in vitro spindle assembly assays leads to monopolar spindles generated by a defect of centrosome separation. In Xenopus cultured cells, pEg2 was confined around the pericentriolar material once centrosomes were duplicated. The centrosome localization does not depend on the presence of microtubules. However, in vitro, the protein binds to taxol-stabilized microtubules independently of its kinase activity. During mitosis the location of the protein changes, in metaphase the kinase localizes on the microtubules at the poles of the mitotic spindle whereas it is not present on astral microtubules. This localization persists until the segregation of the chromosomes is completed. The presence of the kinase on the spindle may reveal another yet unknown function.     

Drosophila Aurora-A is required for centrosome maturation and actin-dependent asymmetric protein localization during mitosis

BACKGROUND: During asymmetric cell division in the Drosophila nervous system, Numb segregates into one of two daughter cells where it is required for the establishment of the correct cell fate. Numb is uniformly cortical in interphase, but in late prophase, the protein concentrates in the cortical area overlying one of two centrosomes in an actin/myosin-dependent manner. What triggers the asymmetric localization of Numb at the onset of mitosis is unclear. RESULTS: We show here that the mitotic kinase Aurora-A is required for the asymmetric localization of Numb. In Drosophila sensory organ precursor (SOP) cells mutant for the aurora-A allele aurA(37), Numb is uniformly localized around the cell cortex during mitosis and segregates into both daughter cells, leading to cell fate transformations in the SOP lineage. aurA(37) mutant cells also fail to recruit Centrosomin (Cnn) and gamma-Tubulin to centrosomes during mitosis, leading to spindle morphology defects. However, Numb still localizes asymmetrically in cnn mutants or after disruption of microtubules, indicating that there are two independent functions for Aurora-A in centrosome maturation and asymmetric protein localization during mitosis. Using photobleaching of a GFP-Aurora fusion protein, we show that two rapidly exchanging pools of Aurora-A are present in the cytoplasm and at the centrosome and might carry out these two functions. CONCLUSIONS: Our results suggest that activation of the Aurora-A kinase at the onset of mitosis is required for the actin-dependent asymmetric localization of Numb. Aurora-A is also involved in centrosome maturation and spindle assembly, indicating that it regulates both actin- and microtubule-dependent processes in mitotic cells.     

Furry Protein Promotes Aurora A-mediated Polo-like Kinase 1 Activation

Bipolar mitotic spindle organization is fundamental to faithful chromosome segregation. Furry (Fry) is an evolutionarily conserved protein implicated in cell division and morphology. In human cells, Fry localizes to centrosomes and spindle microtubules in early mitosis, and depletion of Fry causes multipolar spindle formation. However, it remains unknown how Fry controls bipolar spindle organization. This study demonstrates that Fry binds to polo-like kinase 1 (Plk1) through the polo-box domain of Plk1 in a manner dependent on the cyclin-dependent kinase 1-mediated Fry phosphorylation at Thr-2516. Fry also binds to Aurora A and promotes Plk1 activity by binding to the polo-box domain of Plk1 and by facilitating Aurora A-mediated Plk1 phosphorylation at Thr-210. Depletion of Fry causes centrosome and centriole splitting in mitotic spindles and reduces the kinase activity of Plk1 in mitotic cells and the accumulation of Thr-210-phosphorylated Plk1 at the spindle poles. Our results suggest that Fry plays a crucial role in the structural integrity of mitotic centrosomes and in the maintenance of spindle bipolarity by promoting Plk1 activity at the spindle poles in early mitosis.     

The Caenorhabditis elegans Aurora B kinase AIR-2 phosphorylates and is required for the localization of a BimC kinesin to meiotic and mitotic spindles

BimC kinesins are required for mitotic spindle assembly in a variety of organisms. These proteins are localized to centrosomes, spindle microtubules, and the spindle midzone. We have previously shown that the Caenorhabditis elegans Aurora B kinase AIR-2 is required for the localization of the ZEN-4 kinesin protein to midzone microtubules. To determine whether the association of BimC kinesins with spindle microtubules is also dependent on AIR-2, we examined the expression pattern of BMK-1, a C. elegans BimC kinesin, in wild-type and AIR-2-deficient embryos. BMK-1 is highly expressed in the hermaphrodite gonad and is localized to meiotic spindle microtubules in the newly fertilized embryo. In mitotic embryos, BMK-1 is associated with spindle microtubules from prophase through anaphase and is concentrated at the spindle midzone during anaphase and telophase. In the absence of AIR-2, BMK-1 localization to meiotic and mitotic spindles is greatly reduced. This is not a consequence of loss of ZEN-4 localization because BMK-1 is appropriately localized in ZEN-4-deficient embryos. Furthermore, AIR-2 and BMK-1 directly interact with one another and the C-terminal tail domain of BMK-1 is specifically phosphorylated by AIR-2 in vitro. Together with our previous data, these results suggest that at least one function of the Aurora B kinases is to recruit spindle-associated motor proteins to their sites of action.     

Phosphoregulation of human Mps1 kinase

The dual-specificity protein kinase Mps1 (monopolar spindle 1) is a phosphoprotein required for error-free mitotic progression in eukaryotes. In the present study, we have investigated human Mps1 phosphorylation using combined mass spectrometric, mutational and phosphospecific antibody approaches. We have identified 16 sites of Mps1 autophosphorylation in vitro, several of which are required for catalytic activity after expression in bacteria or in cultured human cells. Using novel phosphospecific antibodies, we show that endogenous Mps1 is phosphorylated on Thr(686) and Ser(821) during mitosis, and demonstrate that phosphorylated Mps1 localizes to the centrosomes of metaphase cells. Taken together, these results reveal the complexity of Mps1 regulation by multi-site phosphorylation, and demonstrate conclusively that phosphorylated Mps1 associates with centrosomes in mitotic human cells.     

The active form of the metabolic sensor AMP-activated protein kinase α (AMPKα) directly binds the mitotic apparatus and travels from centrosomes to the spindle midzone during mitosis and cytokinesis

The metabolic rheostat AMP-activated protein kinase (AMPK) is unexpectedly required for proper cell division and faithful chromosomal segregation during mitosis. Although it is conceptually attractive to assume that AMPK-interpreted microenvironmental bioenergetics may strictly engage cell’s energy status, cell grow, and cell division to avoid that energy stresses trigger cell death, the ultimate framework of AMPK activity towards chromosomal and cytoskeletal mitotic regulation is a question that remains unanswered. We herein reveal that the active form of the α-catalytic AMPK subunit (P-AMPKαThr172) -but not its total form (AMPKα)- transiently associates with several mitotic structures including centrosomes, spindle poles, the central spindle midzone and the midbody throughout all of the mitotic stages and cytokinesis in human cancer-derived epithelial cells. At prophase, P-AMPKαThr172 associates with the two asters of microtubules that begin to nucleate from mature centrosomes. The overlapping localization of P-AMPKαThr172 with the mitotic centrosomal Aurora-A kinase is also apparent on the microtubules near the spindle poles in metaphase and in early anaphase. This Aurora A-like centrosomal localization of P-AMPKαThr172 cannot be detected following chromatid separation following anaphase-telophase transition. Rather, toward the end of anaphase and in telophase P-AMPKαThr172 reactivity exhibited a similar but not identical localization to that occupied by the bona fide chromosomal passenger proteins INCENCP and Aurora-B. This localization of P-AMPKαThr172 at the central spindle and midbody persisted during the furrowing process and, at the completion of telophase, a prominent staining of P-AMPKαThr172 as doublet was apparent on either side of the midbody within the intercellular cytokinetic bridge. An identical mitotic geography of P-AMPKαThr172 was observed in cancer cells lacking the AMPK kinase LKB1, in non-cancerous human epithelial cells, and in mouse fibroblasts. The active form of AMPKα bound to the mitotic apparatus may physically tether the bioenergetic state of a cell to the four-dimensional regulation of the chromosomal and cytoskeletal mitotic events, thus suggesting a putative cytokinetic suppressor function. In this newly discovered scenario, we suggest a primordial mitotic role for the α catalytic AMPK subunit in the eukaryotic evolutionary process as it may ensure, at the cell level, an exquisite coordination between sensing of energy resources and the fundamental biological process of genome division.     

Inhibition of nuclear import of LIMK2 in endothelial cells by protein kinase C-dependent phosphorylation at Ser-283

LIM kinases (LIMKs) are mainly in the cytoplasm and regulate actin dynamics through cofilin phosphorylation. Recently, it has been reported that nuclear localization of LIMKs can mediate suppression of cyclin D1 expression. Using immunofluorescence monitoring of enhanced green fluorescent protein-tagged LIMK2 in combination with photobleaching techniques and leptomycin B treatment, we demonstrate that LIMK2 shuttles between the cytoplasm and the nucleus in endothelial cells. Sequence analysis predicted two PKC phosphorylation sites in LIMK2 but not in LIMK1. One site at Ser-283 is present between the PDZ and the kinase domain, and the other site at Thr-494 is within the kinase domain. Activation of PKC by phorbol ester treatment of endothelial cells stimulated LIMK2 phosphorylation at Ser-283 and inhibited nuclear import of LIMK2 and the PDZ kinase construct of LIMK2 (amino acids 142-638) but not of LIMK1. The PKC-delta isoform phosphorylated LIMK2 at Ser-283 in vitro. Mutational analysis indicated that LIMK2 phosphorylation at Ser-283 but not Thr-494 was functional. Serum stimulation of endothelial cells also inhibited nuclear import of PDZK-LIMK2 by protein kinase C-dependent phosphorylation of Ser-283. Our study shows that phorbol ester and serum stimulation of endothelial cells inhibit nuclear import of LIMK2 but not LIMK1. This effect was dependent on PKC-delta-mediated phosphorylation of Ser-283. Since phorbol ester enhanced cyclin D1 expression and subsequent G1-to-S-phase transition of endothelial cells, we suggest that the PKC-mediated exclusion of LIMK2 from the nucleus might be a mechanism to relieve suppression of cyclin D1 expression by LIMK2.     

Activation of LIM kinases by myotonic dystrophy kinase-related Cdc42-binding kinase alpha

LIM kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through cofilin phosphorylation downstream of distinct Rho family GTPases. Pak1 and ROCK, respectively, activate LIMK1 and LIMK2 downstream of Rac and Rho; however, an effector protein kinase for LIMKs downstream of Cdc42 remains to be defined. We now report evidence that LIMK1 and LIMK2 activities toward cofilin phosphorylation are stimulated in cells by the co-expression of myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha), an effector protein kinase of Cdc42. In vitro, MRCKalpha phosphorylated the protein kinase domain of LIM kinases, and the site in LIMK2 phosphorylated by MRCKalpha proved to be threonine 505 within the activation segment. Expression of MRCKalpha induced phosphorylation of actin depolymerizing factor (ADF)/cofilin in cells, whereas MRCKalpha-induced ADF/cofilin phosphorylation was inhibited by the co-expression with the protein kinase-deficient form of LIM kinases. These results indicate that MRCKalpha phosphorylates and activates LIM kinases downstream of Cdc42, which in turn regulates the actin cytoskeletal reorganization through the phosphorylation and inactivation of ADF/cofilin.     

Phosphorylation-dependent regulation of unique nuclear and nucleolar localization signals of LIM kinase 2 in endothelial cells

LIM kinases (LIMKs) regulate actin dynamics through cofilin phosphorylation and also have a function in the nucleus. Recently we have shown that LIMK2 shuttles between cytoplasm and nucleus in endothelial cells and that nuclear import is inhibited by protein kinase C-mediated phosphorylation of Ser-283. Here we aimed to identify the structural features of LIMK2 responsible for nuclear import. We found that the kinase domain of LIMK2 is localized exclusively in the nucleus and, in contrast to the kinase domain of LIMK1, it accumulated in the nucleolus. Through site-directed mutagenesis, we identified the basic amino acid-rich motif KKRTLRKNDRKKR (amino acids 491-503) as the functional nuclear and nucleolar localization signal of LIMK2. After fusing this motif to enhanced green fluorescent protein, the fusion protein localized exclusively in the nucleus and nucleolus. Mutagenesis studies showed that phosphorylation of Thr-494, a putative protein kinase C phosphorylation site identified within the nuclear localization signal, inhibits nuclear import of the enhanced green fluorescent protein-PDZ kinase domain of LIMK2. After inhibiting nuclear export with leptomycin B, phosphorylation of either Ser-283 or Thr-494 reduced the nuclear import of LIMK2. Phosphorylation of both Ser-283 and Thr-494 sites inhibited nuclear import completely. Our findings identify a unique basic amino acid-rich motif (amino acids 491-503) in LIMK2 which is not present in LIMK1 that serves to target the protein not only to the nucleus but also to the nucleolus. Phosphorylation of Thr-494 within this motif negatively regulates nuclear import of LIMK2.