A specific interaction between the telomeric protein Pin2/TRF1 and the mitotic spindle

Pin2/TRF1 was independently identified as a telomeric DNA binding protein (TRF1) [1] and as a protein (Pin2) that can bind the mitotic kinase NIMA and suppress its ability to induce mitotic catastrophe [2, 3]. Pin2/TRF1 has been shown to bind telomeric DNA as a dimer [3-7] and to negatively regulate telomere length [8-11]. Interestingly, Pin2/TRF1 levels are regulated during the cell cycle, being increased in late G2 and mitosis and degraded as cells exit from mitosis [3]. Furthermore, overexpression of Pin2/TRF1 induces mitotic entry and then apoptosis [12]. This Pin2/TRF1 activity can be significantly potentiated by the microtubule-disrupting agent nocodazole [12] but is suppressed by phosphorylation of Pin2/TRF1 by ATM; this negative regulation is important for preventing apoptosis upon DNA damage [13]. These results suggest a role for Pin2/TRF1 in mitosis. However, nothing is known about how Pin2/TRF1 is involved in mitotic progression. Here, we describe a surprising physical interaction between Pin2/TRF1 and microtubules in a cell cycle-specific manner. Both expressed and endogenous Pin2/TRF1 proteins were localized to the mitotic spindle during mitosis. Furthermore, Pin2/TRF1 directly bound microtubules via its C-terminal domain. Moreover, Pin2/TRF1 also promoted microtubule polymerization in vitro. These results demonstrate for the first time a specific interaction between Pin2/TRF1 and microtubules in a mitosis-specific manner, and they suggest a new role for Pin2/TRF1 in modulating the function of microtubules during mitosis.     

Involvement of the telomeric protein Pin2/TRF1 in the regulation of the mitotic spindle

Pin2/TRF1 was independently identified as a telomeric DNA-binding protein (TRF1) that regulates telomere length, and as a protein (Pin2) that can bind the mitotic kinase NIMA and suppress its lethal phenotype. We have previously demonstrated that Pin2/TRF1 levels are cell cycle-regulated and its overexpression induces mitotic arrest and then apoptosis. This Pin2/TRF1 activity can be potentiated by microtubule-disrupting agents, but suppressed by phosphorylation of Pin2/TRF1 by ATM; this negative regulation is critical in mediating for many, but not all, ATM-dependent phenotypes. Interestingly, Pin2/TRF1 specifically localizes to mitotic spindles in mitotic cells and affects the microtubule polymerization in vitro. These results suggest a role of Pin2/TRF1 in mitosis. However, nothing is known about whether Pin2/TRF1 affects the spindle function in mitotic progression. Here we characterized a new Pin2/TRF1-interacting protein, EB1, that was originally identified in our yeast two-hybrid screen. Pin2/TRF1 bound EB1 both in vitro and in vivo and they also co-localize at the mitotic spindle in cells. Furthermore, EB1 inhibits the ability of Pin2/TRF1 to promote microtubule polymerization in vitro. Given that EB1 is a microtubule plus end-binding protein, these results further confirm a specific interaction between Pin2/TRF1 and the mitotic spindle. More importantly, we have shown that inhibition of Pin2/TRF1 in ataxia-telangiectasia cells is able to fully restore their mitotic spindle defect in response to microtubule disruption, demonstrating for the first time a functional involvement of Pin2/TRF1 in mitotic spindle regulation.     

Binding of phosphatase inhibitor-2 to prolyl isomerase Pin1 modifies specificity for mitotic phosphoproteins

Inhibitor-2 (I-2) is the most ancient protein that selectively recognizes type-1 protein phosphatase and is phosphorylated by CDK1-cyclinB during mitosis at Thr72 in a conserved PXTP site. Pin1 is a peptide prolyl cis/trans isomerase conserved among eukaryotes that specifically reacts with proteins phosphorylated at Ser/Thr-Pro sites. We tested phospho-T72-I-2 as a substrate for Pin1 and discovered that unphosphorylated I-2 bound Pin1 with micromolar affinity and phosphorylation of the PXTP site or truncation of I-2 reduced binding 10-fold. Ectopic Pin1 coprecipitated endogenous I-2 and ectopic I-2 coprecipitated endogenous Pin1, but only in the absence of detergents, which may account for the interaction not being detected previously. Endogenous I-2 and Pin1 colocalized in HeLa cells and showed nuclear-cytoplasmic redistribution in response to cell density, suggestive of their association in living cells. Recombinant Pin1 binding to different phosphoproteins in mitotic cell extracts was modulated by I-2, and binding to individual mitotic phosphoproteins was increased, decreased or unaffected by I-2, showing that I-2 allosterically modifies Pin1 specificity. This was confirmed by mutation of Ser16 to Ala in the Pin1 WW domain that eliminated I-2 binding and abrogated I-2 effects on Pin1 binding to different phosphoproteins. A S16E mutation to mimic Pin1 phosphorylation restored binding to both I-2 and phospho-T72-I-2, indicating that phosphorylation of both proteins governs their interaction. The results reveal a novel function for I-2, and suggest phosphorylation-dependent regulation of Pin1 specificity during entry and exit of mitosis, in other phases of the cell cycle, and in multiple cell signaling processes.     

Chromosomal passengers: the four-dimensional regulation of mitotic events

Chromosomal passengers are proteins that are involved in coordinating the chromosomal and cytoskeletal events of mitosis. The passengers are present in cells as a complex with at least four members: Aurora B, a protein kinase; inner centromeric protein, an activation and targeting subunit; Survivin (function unknown) and Borealin (function also unknown). The kinase is activated at the onset of mitosis, at least partly accomplished by regulation of the levels of its constituents. As mitosis progresses, the kinase complex moves to a highly choreographed series of locations in the mitotic cell, activating key substrates at precise locations and specific times. Functions that require chromosomal passenger activity include chromatin modification (phosphorylation of histone H3), correction of kinetochore attachment errors, aspects of the spindle assembly checkpoint, assembly of a stable bipolar spindle and the completion of cytokinesis. The chromosomal passenger complex provides an essential mechanism for mitotic regulation.     

The Survivin-Crm1 interaction is essential for chromosomal passenger complex localization and function

The chromosomal passenger complex (CPC) of Aurora-B, Borealin, INCENP (inner centromere protein) and Survivin coordinates essential chromosomal and cytoskeletal events during mitosis. Here, we show that the nuclear export receptor Crm1 is crucially involved in tethering the CPC to the centromere by interacting with a leucine-rich nuclear export signal (NES), evolutionarily conserved in all mammalian Survivin proteins. We show that inhibition of the Survivin-Crm1 interaction by treatment with leptomycin B or by RNA-interference-mediated Crm1 depletion prevents centromeric targeting of Survivin. The genetic inactivation of the Survivin-Crm1 interaction by mutation of the NES affects the correct localization and function of Survivin and the CPC during mitosis. By contrast, CPC assembly does not seem to require the Survivin-Crm1 interaction. Our report shows the functional significance of the Survivin-Crm1 interface and provides a novel link between the mitotic effector Crm1 and the CPC.     

Polo-like kinase 1-mediated phosphorylation stabilizes Pin1 by inhibiting its ubiquitination in human cells

The Polo-like kinase 1 (Plk1) is a key regulator of mitosis. It is reported that the human peptidyl-prolyl cis/trans-isomerase Pin1 binds to Plk1 from mitotic cell extracts in vitro. Here we demonstrate that Ser-65 in Pin1 is the major site for Plk1-specific phosphorylation, and the polo-box domain of Plk1 is required for this phosphorylation. Interestingly, the phosphorylation of Pin1 by Plk1 does not affect its isomerase activity but rather is linked to its protein stability. Pin1 is ubiquitinated in HeLa S3 cells, and substitution of Glu for Ser-65 reduces the ubiquitination of Pin1. Furthermore, inhibition of Plk1 activity by expression of a dominant negative form of Plk1 or by transfection of small interfering RNA targeted to Plk1 enhances the ubiquitination of Pin1 and subsequently reduces the amount of Pin1 in human cancer cells. Since previous reports suggested that Plk1 is a substrate of Pin1, our work adds a new dimension to this interaction of two important mitotic regulators.     

IGF-1 induces Pin1 expression in promoting cell cycle S-phase entry.

Insulin-like growth factor I (IGF-1) is a well-established mitogen to many different cell types and is implicated in progression of a number of human cancers, notably breast cancer. The prolyl isomerase Pin1 plays an important role in cell cycle regulation through its specific interaction with proteins that are phosphorylated at Ser/Thr-Pro motifs. Pin1 knockout mice appear to have relatively normal development yet the Pin1(-/-)mouse embryo fibroblast (MEF) cells are defective in re-entering cell cycle in response to serum stimulation after G0 arrest. Here, we report that Pin1(-/-) MEF cells display a delayed cell cycle S-phase entry in response to IGF stimulation and that IGF-1 induces Pin1 protein expression which correlates with the induction of cyclin D1 and RB phosphorylation in human breast cancer cells. The induction of Pin1 by IGF-1 is mediated via the phosphatidylinositol 3-kinase as well as the MAP kinase pathways. Treatment of PI3K inhibitor LY294002 and the MAP kinase inhibitor PD098059, but not p38 inhibitor SB203580, effectively blocks IGF-1-induced upregulation of Pin1, cyclin D1 and RB phosphorylation. Furthermore, we found that Cyclin D1 expression and RB phosphorylation are dramatically decreased in Pin1(-/-) MEF cells. Reintroducing a recombinant adenovirus encoding Pin1 into Pin1(-/-) MEF cells restores the expression of cyclin D1 and RB phosphorylation. Thus, these data suggest that the mitogenic function of IGF-1 is at least partially linked to the induction of Pin1, which in turn stimulates cyclin D1 expression and RB phosphorylation, therefore contributing to G0/G1-S transition.     

Telomeric protein Pin2/TRF1 as an important ATM target in response to double strand DNA breaks

ATM mutations are responsible for the genetic disease ataxia-telangiectasia (A-T). ATM encodes a protein kinase that is activated by ionizing radiation-induced double strand DNA breaks. Cells derived from A-T patients show many abnormalities, including accelerated telomere loss and hypersensitivity to ionizing radiation; they enter into mitosis and apoptosis after DNA damage. Pin2 was originally identified as a protein involved in G(2)/M regulation and is almost identical to TRF1, a telomeric protein that negatively regulates telomere elongation. Pin2 and TRF1, probably encoded by the same gene, PIN2/TRF1, are regulated during the cell cycle. Furthermore, up-regulation of Pin2 or TRF1 induces mitotic entry and apoptosis, a phenotype similar to that of A-T cells after DNA damage. These results suggest that ATM may regulate the function of Pin2/TRF1, but their exact relationship remains unknown. Here we show that Pin2/TRF1 coimmunoprecipitated with ATM, and its phosphorylation was increased in an ATM-dependent manner by ionizing DNA damage. Furthermore, activated ATM directly phosphorylated Pin2/TRF1 preferentially on the conserved Ser(219)-Gln site in vitro and in vivo. The biological significance of this phosphorylation is substantiated by functional analyses of the phosphorylation site mutants. Although expression of Pin2 and its mutants has no detectable effect on telomere length in transient transfection, a Pin2 mutant refractory to ATM phosphorylation on Ser(219) potently induces mitotic entry and apoptosis and increases radiation hypersensitivity of A-T cells. In contrast, Pin2 mutants mimicking ATM phosphorylation on Ser(219) completely fail to induce apoptosis and also reduce radiation hypersensitivity of A-T cells. Interestingly, the phenotype of the phosphorylation-mimicking mutants is the same as that which resulted from inhibition of endogenous Pin2/TRF1 in A-T cells by its dominant-negative mutants. These results demonstrate for the first time that ATM interacts with and phosphorylates Pin2/TRF1 and suggest that Pin2/TRF1 may be involved in the cellular response to double strand DNA breaks.     

Prolyl-peptidyl isomerase,Pin1, phosphorylation is compromised in association with the expression of the HFE polymorphic allele,H63D

There is substantial interest in HFE gene variants as putative risk factors in neurodegenerative diseases such as Alzheimer disease (AD). Previous studies in cell models have shown the H63D HFE variant to result in increased cellular iron, oxidative stress, glutamate dyshomeostasis, and an increase in tau phosphorylation; all processes thought to contribute to AD pathology. Pin1 is a prolyl-peptidyl cis/trans isomerase that can regulate the dephosphorylation of the amyloid and tau proteins. Hyperphosphorylation of these later proteins is implicated in the pathogenesis of AD and Pin1 levels are reportedly decreased in AD brains. Because of the relationship between Pin1 loss of function by oxidative stress and the increase in oxidative stress in cells with the H63D polymorphism it was logical to interrogate a relationship between Pin1 and HFE status. To test our hypothesis that H63D HFE would be associated with less Pin1 activity, we utilized stably transfected human neuroblastoma SH-SY5Y cell lines expressing the different HFE polymorphisms. Under resting conditions, total Pin1 levels were unchanged between the wild type and H63D HFE cells, yet there was a significant increase in phosphorylation of Pin1 at its serine 16 residue suggesting a loss of Pin1 activity in H63D variant cells. To evaluate whether cellular iron status could influence Pin1, we treated the WT HFE cells with exogenous iron and found that Pin1 phosphorylation increased with increasing levels of iron. Iron exposure to H63D variant cells did not impact Pin1 phosphorylation beyond that already seen suggesting a ceiling effect. Because HFE H63D cells have been shown to have more oxidative stress, the cells were treated with the antioxidant Trolox which resulted in a decrease in Pin1 phosphorylation in H63D cells with no change in WT HFE cells. In a mouse model carrying the mouse equivalent of the H63D allele, there was an increase in the phosphorylation status of Pin1 providing in vivo evidence for our findings in the cell culture model. Thus, we have shown another cellular mechanism that HFE polymorphisms influence; further supporting their role as neurodegenerative disease modifiers.     

Role of Pin2/TRF1 in telomere maintenance and cell cycle control

Telomeres are specialized structures found at the extreme ends of chromosomes, which have many functions, including preserving genomic stability, maintaining cell proliferative capacity, and blocking the activation of DNA-damage cell cycle checkpoints. Deregulation of telomere length has been implicated in cancer and ageing. Telomere maintenance is tightly regulated by telomerase and many other telomere-associated proteins and is also closely linked to cell cycle control, especially mitotic regulation. However, little is known about the identity and function of the signaling molecules connecting telomere maintenance and cell cycle control. Pin2/TRF1 was originally identified as a protein bound to telomeric DNA (TRF1) and as a protein involved in mitotic regulation (Pin2). Pin2/TRF1 negatively regulates telomere length and importantly, its function is tightly regulated during the cell cycle, acting as an important regulator of mitosis. Recent identification of many Pin2/TRF1 upstream regulators and downstream targets has provided important clues to understanding the dual roles of Pin2/TRF1 in telomere maintenance and cell cycle control. These results have led us to propose that Pin2/TRF1 functions as a key molecule in connecting telomere maintenance and cell cycle control.     

Mitotic Regulation of SEPT9 Protein by Cyclin-dependent Kinase 1 (Cdk1) and Pin1 Protein Is Important for the Completion of Cytokinesis

Precise cell division is essential for multicellular development, and defects in this process have been linked to cancer. Septins are a family of proteins that are required for mammalian cell division, but their function and mode of regulation during this process are poorly understood. Here, we demonstrate that cyclin-dependent kinase 1 (Cdk1) phosphorylates septin 9 (SEPT9) upon mitotic entry, and this phosphorylation controls association with the proline isomerase, Pin1. Both SEPT9 and Pin1 are critical for mediating the final separation of daughter cells. Expression of mutant SEPT9 that is defective in Pin1 binding was unable to rescue cytokinesis defects caused by SEPT9 depletion but rather induced dominant-negative defects in cytokinesis. However, unlike SEPT9 depletion, Pin1 was not required for the accumulation of the exocyst complex at the midbody. These results suggest that SEPT9 plays multiple roles in abscission, one of which is regulated by the action of Cdk1 and Pin1.     

The mitotic regulator Survivin binds as a monomer to its functional interactor Borealin

Survivin is a member of the IAP (inhibitor of apoptosis) protein family, defined in part by the presence of a zinc-binding baculoviral inhibitory repeat (BIR) domain. Most BIR domains bind short sequences beginning with alanine, and in this manner, they recognize and block the action of key targets in apoptotic pathways. However, Survivin binds only very weakly to typical IAP ligands. Unique features of Survivin are the long C-terminal helix following the BIR domain and a short segment (linking the helix and BIR domains) that mediates Survivin homodimerization. Despite this detailed knowledge of the structure of Survivin itself, there is a current lack of understanding about how Survivin recognizes cellular binding partners, and consequently, many questions about Survivin function remain unanswered. We determined two co-crystal structures of Survivin and a minimal binding fragment from the chromosomal passenger protein Borealin, a well validated functional interactor. The interaction between Survivin and Borealin involves extensive packing between the long C-terminal helix of Survivin and a long Borealin helix. Surprisingly, an additional important interaction occurs between the Survivin homodimerization interface and a short segment of Borealin. This segment both structurally mimics and displaces one Survivin monomer. The relevance of this unexpected interaction was tested by mutagenesis of two key Borealin residues. Mutant Borealin introduced into HeLa cells failed to localize properly during mitosis and also caused mislocalization of other chromosomal passenger proteins. This suggests that the mutant is dominant-negative and confirms the functional importance of the interaction surface identified in the crystal structures.     

The F-box protein FBX4 targets PIN2/TRF1 for ubiquitin-mediated degradation and regulates telomere maintenance

Pin2/TRF1 was identified previously as both a protein (TRF1) that binds to telomeric DNA repeats and as a protein (Pin2) that associates with the kinase NIMA and suppresses its mitosis inducing activity. Pin2/TRF1 negatively regulates telomere length and also plays a critical role in cell cycle checkpoint control. Pin2/TRF1 is down-regulated in many human cancers and may be degraded by the ubiquitin-proteasome pathway, but components of the pathway involved in Pin2/TRF1 turnover have not been elucidated. By using the two-hybrid system, we recently identified Pin2/TRF1-interacting proteins, PinX1-4, and we demonstrated that PinX1 is a conserved telomerase inhibitor and a putative tumor suppressor. Here we report the characterization of PinX3. PinX3 was later found to be identical to Fbx4, a member of the F-box family of proteins, which function as substrate-specific adaptors of Cul1-based ubiquitin ligases. Fbx4 interacts with both Pin2 and TRF1 isoforms and promotes their ubiquitination in vitro and in vivo. Moreover, overexpression of Fbx4 reduces endogenous Pin2/TRF1 protein levels and causes progressive telomere elongation in human cells. In contrast, inhibition of Fbx4 by RNA interference stabilizes Pin2/TRF1 and promotes telomere shortening, thereby impairing cell growth. These results demonstrate that Fbx4 is a central regulator of Pin2/TRF1 protein abundance and that alterations in the stability of Pin2/TRF1 can have a dramatic impact on telomere length. Thus, Fbx4 may play a critical role in telomere maintenance.     

Interactions between protein kinase CK2 and Pin1. Evidence for phosphorylation-dependent interactions

The peptidyl-prolyl isomerase Pin1 interacts in a phosphorylation-dependent manner with several proteins involved in cell cycle events. In this study, we demonstrate that Pin1 interacts with protein kinase CK2, an enzyme that generally exists in tetrameric complexes composed of two catalytic CK2 alpha and/or CK2 alpha' subunits together with two regulatory CK2 beta subunits. Our results indicate that Pin1 can interact with CK2 complexes that contain CK2 alpha. Furthermore, Pin1 can interact directly with the C-terminal domain of CK2 alpha that contains residues that are phosphorylated in vitro by p34(Cdc2) and in mitotic cells. Substitution of the phosphorylation sites of CK2 alpha with alanines resulted in decreased interactions between Pin1 and CK2. The other catalytic isoform of CK2, designated CK2 alpha', is not phosphorylated in mitotic cells and does not interact with Pin1, but a chimeric protein consisting of CK2 alpha' with the C terminus of CK2 alpha was phosphorylated in mitotic cells and interacts with Pin1, further implicating the phosphorylation sites in the interaction. In vitro, Pin1 inhibits the phosphorylation of Thr-1342 on human topoisomerase II alpha by CK2. Topoisomerase II alpha also interacts with Pin1 suggesting that the effect of Pin1 on the phosphorylation of Thr-1342 could result from its interactions with CK2 and/or topoisomerase II alpha. As compared with wild-type Pin1, isomerase-deficient and WW domain-deficient mutants of Pin1 are impaired in their ability to interact with CK2 and to inhibit the CK2-catalyzed phosphorylation of topoisomerase II alpha. Collectively, these results indicate that Pin1 and CK2 alpha interact and suggest a possible role for Pin1 in the regulation of topoisomerase II alpha. Furthermore, these results provide new insights into the functional role of the mitotic phosphorylation of CK2 and provide a new mechanism for selectively regulating the ability of CK2 to phosphorylate one of its mitotic targets.     

Cep55 stabilization is required for normal execution of cytokinesis

Cep55 is a mitotic phosphoprotein that plays an important role in cytokinesis, the final stage of cell division during which physical separation of the two daughter cells is accomplished. We recently demonstrated that the peptidyl-prolyl isomerase Pin1 regulates this cell cycle event by enhancing the Plk1-dependent phosphorylation of Cep55. We show here that Cep55 is stabilized post-translationally during mitosis and that siRNA-mediated knockdown of Pin1 prevents this stabilization. Consistent with this, Cep55 is unstable in Pin1 knockout mouse embryonic fibroblasts. Moreover, mutation of the Pin1 binding sites in Cep55 reduces its stability during mitosis. Mutation of the Plk1 phosphorylation site also lowers Cep55 stability, whereas overexpression of Plk1 increases Cep55 levels, in keeping with Pin1 regulating Plk1-mediated phosphorylation of Cep55. Importantly, expression of wild-type Cep55 at levels similar to that of the phosphorylation mutants only partially reverts the cytokinesis defect induced by depletion of Cep55, indicating that inadequate levels of Cep55 prevent proper execution of cytokinesis. Taken together, these data provide more insight into the regulation of the final stages of cell division. As cytokinesis defects can cause chromosomal instability, knowledge about the processes that regulate normal cytokinesis adds to our understanding of events that lead to tumorigenesis.     

A critical role for Pin2/TRF1 in ATM-dependent regulation. Inhibition of Pin2/TRF1 function complements telomere shortening, radiosensitivity, and the G(2)/M checkpoint defect of ataxia-telangiectasia cells.

Cells derived from patients with the human genetic disorder ataxia-telangiectasia (A-T) display many abnormalities, including telomere shortening, premature senescence, and defects in the activation of S phase and G(2)/M checkpoints in response to double-strand DNA breaks induced by ionizing radiation. We have previously demonstrated that one of the ATM substrates is Pin2/TRF1, a telomeric protein that binds the potent telomerase inhibitor PinX1, negatively regulates telomere elongation, and specifically affects mitotic progression. Following DNA damage, ATM phosphorylates Pin2/TRF1 and suppresses its ability to induce abortive mitosis and apoptosis (Kishi, S., Zhou, X. Z., Nakamura, N., Ziv, Y., Khoo, C., Hill, D. E., Shiloh, Y., and Lu, K. P. (2001) J. Biol. Chem. 276, 29282-29291). However, the functional importance of Pin2/TRF1 in mediating ATM-dependent regulation remains to be established. To address this question, we directly inhibited the function of endogenous Pin2/TRF1 in A-T cells by stable expression of two different dominant-negative Pin2/TRF1 mutants and then examined their effects on telomere length and DNA damage response. Both the Pin2/TRF1 mutants increased telomere length in A-T cells, as shown in other cells. Surprisingly, both the Pin2/TRF1 mutants reduced radiosensitivity and complemented the G(2)/M checkpoint defect without inhibiting Cdc2 activity in A-T cells. In contrast, neither of the Pin2/TRF1 mutants corrected the S phase checkpoint defect in the same cells. These results indicate that inhibition of Pin2/TRF1 in A-T cells is able to bypass the requirement for ATM in specifically restoring telomere shortening, the G(2)/M checkpoint defect, and radiosensitivity and demonstrate a critical role for Pin2/TRF1 in the ATM-dependent regulation of telomeres and DNA damage response.     

Interaction and feedback regulation between STK15/BTAK/Aurora-A kinase and protein phosphatase 1 through mitotic cell division cycle

STK15 is an Aurora/Ipl-1 related serine/threonine kinase that is associated with centrosomes and induces aneuploidy when overexpressed in mammalian cells. It is well known that phosphorylation and dephosphorylation of kinases are important for regulation of their activity. But mechanisms by which STK15 activity is regulated have not been elucidated. We report that STK15 contains two functional binding sites for protein phosphatase type 1 (PP1), and the binding of these proteins is cell cycle-regulated peaking at mitosis. Activated STK15 at mitosis phosphorylates PP1 and inhibits PP1 activity in vitro. In vivo, PP1 activity co-immunoprecipitated with STK15 is also reduced. These data indicate that STK15 inhibits PP1 activity during mitosis. Also, PP1 is shown to dephosphorylate active STK15 and abolish its activity in vitro. Furthermore, we show that non-binding mutants of STK15 for PP1 are superphosphorylated, but their kinase activities are markedly reduced. Cells transfected with these non-binding mutants manifest aberrant chromosome alignment during mitosis. Our results suggest that a feedback regulation through phosphorylation/dephosphorylation events between STK15 kinase and PP1 phosphatase operates through the cell cycle. Deregulation of this balance may contribute to anomalous segregation of chromosomes during mitotic progression of cancer cells.