Identification of a novel kinesin-related protein, KRMP1, as a target for mitotic peptidyl-prolyl isomerase Pin1

Mitosis utilizes a number of kinesin-related proteins (KRPs). Here we report the identification of a novel KRP termed KRMP1, which has a deduced 1780-amino acid sequence composed of ternary domains. The amino-terminal head domain is most similar to the kinesin motor domain of the MKLP-1 subfamily and has an intrinsic ATPase activity that is diminished by substituting the consensus Lys-168 with Arg. The central stalk domain is predicted to form a long alpha-helical coiled-coil, and can interact with each other in vivo. An in vivo labeling experiment revealed that KRMP1 is phosphorylated, and we also found that the region within the tail domain containing Thr-1604 as the cdc2 kinase phosphorylation site differs from the bimC box conserved in the bimC subfamily of KRPs. Immunofluorescence analysis showed that endogenous KRMP1 was localized predominantly to the cytoplasm during interphase and dispersed throughout the cell during mitosis. Consistent with this finding, overexpressed KRMP1 was detected in a complicated nuclear or cytoplasmic pattern reflecting multiple nuclear localization/export signals. Furthermore, KRMP1 interacted with the mitotic peptidyl-prolyl isomerase Pin1 in vivo, and an in vitro interaction was detected between the tail domain of KRMP1 and the WW domain of Pin1. Overexpression of KRMP1 caused COS-7 cells to arrest at G(2)-M, and co-expression of Pin1 reversed this effect, indicating their physiological interaction. Together, our results suggest that KRMP1 is a mitotic target regulated by Pin1 and vice versa.     

The prolyl isomerase Pin1 functions in mitotic chromosome condensation

The prolyl isomerase Pin1 plays important roles in numerous cellular processes. Here we provide evidence that Pin1 has an important function in chromosome condensation during mitosis. We first demonstrate that the interaction of Pin1 with chromatin is greatly elevated in G2/M phase and that this correlates with the presence on chromosomes of several mitotic phosphoproteins, especially topoisomerase (Topo) IIalpha. Inducible overexpression of Pin1 was shown to result in higher M phase-specific phosphorylation, while downregulation of Pin1 by siRNA treatment reduced phosphorylation of TopoIIalpha and other mitotic proteins. Furthermore, immunodepletion of Pin1 from mitotic cell extracts prevented such extracts from inducing chromosome condensation when added to S phase nuclei. Indeed, purified Pin1 and cdc2/cyclin B kinase were by themselves sufficient to induce condensation. This reflects the ability of Pin1 to increase TopoIIalpha phosphorylation by cdc2/cyclin B in vitro, which in turn dramatically increased formation of a TopoIIalpha/Pin1/DNA complex.     

The peptidyl-prolyl isomerase Pin1 regulates granulocyte-macrophage colony-stimulating factor mRNA stability in T lymphocytes

Cytokine production is associated with both the normal and pathologic inflammatory response to injury. Previous studies have shown that the immunosuppressants cyclosporin A or FK506, which interact with the peptidyl-propyl isomerases cyclophilin A and FK506-binding protein (FKBP12), respectively, block cytokine expression. A third member of the peptidyl-propyl isomerase family, Pin1 is expressed by immune and other cells. Pin1 has been implicated in cell cycle progression, is overexpressed in human tumors, and may rescue neurons from tau-associated degeneration. However, the role of Pin1 in the immune system remains largely unknown. In this study, we analyze the role of Pin1 in GM-CSF expression by human PBMC and CD4+ lymphocytes. We show that Pin1 isomerase activity is necessary for activation-dependent, GM-CSF mRNA stabilization, accumulation, and protein secretion, but not non-AU-rich elements containing cytokine mRNAs, including TGF-beta and IL-4. Mechanistically, Pin1 mediated the association of the AU-rich element-binding protein, AUF1, with GM-CSF mRNA, which determined the rate of decay by the exosome.     

Molecular dynamics simulations of wild type and mutant of Pin1 peptidyl-prolyl isomerase

Pin1 catalyses the intrinsically slow process of cis-trans isomerisation and has been identified as a possible drug target in many diseases. Recently, the wild type (WT) and the Cys113Asp mutant of the Pin1 peptidyl-prolyl isomerase (PPIase) domain were determined by nuclear magnetic resonance. In this article, the WT and Cys113Asp mutant of PPIase domain are studied by molecular dynamics simulations. The structural stability analysis shows that the Cys113Asp mutation leads to the higher fluctuation of hydrophobic core in PPIase domain. The intrinsic correlated motions are important for the catalytic function of Pin1, whereas the Cys113Asp mutant system loses pivotal dynamical properties and develops wider conformational states than those in WT system. The intramolecular hydrogen bonds play crucial roles in the structural stability of PPIase domain. The mutated residue Asp113 attracts the side chain of His59 in the Cys113Asp system, which unbalances the internal interactions inside the catalytic tetrad. Meanwhile, the conformational changes of PPIase domain affect the side chain orientations of Lys63 and Arg69, which limit their binding with substrates. The Cys113Asp mutation destabilises the whole binding region of Pin1 PPIase domain, so the catalysis activity is severely reduced. These results are consistent with experimental studies and may help to understand the isomerisation mechanisms of Pin1.     

Inactivating Cdc25, mitotic style

Mitotic entry and exit require activation and inactivation of the Cdk1-cyclin B kinase complex, respectively. The Cdc25 protein phosphatase family activates Cdk1-cyclin B at the G2/M transition by removing inhibitory phosphate groups. Cdc25 family members, held inactive during interphase, are activated during mitotic progression in an amplification loop involving Cdk1-cyclin B. While Cdc25 activation at the G2/M transition is required for the timely initiation of mitosis, recent evidence suggests that the inactivation of Cdc25 in late mitosis may play a role in supporting Cdk1-cyclin B inactivation. Here, we discuss the mechanisms of Cdc25 regulation and how they pertain to both mitotic entry and exit.     

Functionally important residues in the peptidyl-prolyl isomerase Pin1 revealed by unigenic evolution

Pin1 is a phosphorylation-dependent member of the parvulin family of peptidyl-prolyl isomerases exhibiting functional conservation between yeast and man. To perform an unbiased analysis of the regions of Pin1 essential for its functions, we generated libraries of randomly mutated forms of the human Pin1 cDNA and identified functional Pin1 alleles by their ability to complement the Pin1 homolog Ess1 in Saccharomyces cerevisiae. We isolated an extensive collection of functional mutant Pin1 clones harboring a total of 356 amino acid substitutions. Surprisingly, many residues previously thought to be critical in Pin1 were found to be altered in this collection of functional mutants. In fact, only 17 residues were completely conserved in these mutants and in Pin1 sequences from other eukaryotic organisms, with only two of these conserved residues located within the WW domain of Pin1. Examination of invariant residues provided new insights regarding a phosphate-binding loop that distinguishes a phosphorylation-dependent peptidyl-prolyl isomerase such as Pin1 from other parvulins. In addition, these studies led to an investigation of residues involved in catalysis including C113 that was previously implicated as the catalytic nucleophile. We demonstrate that substitution of C113 with D does not compromise Pin1 function in vivo nor does this substitution abolish catalytic activity in purified recombinant Pin1. These findings are consistent with the prospect that the function of residue 113 may not be that of a nucleophile, thus raising questions about the model of nucleophilic catalysis. Accordingly, an alternative catalytic mechanism for Pin1 is postulated.     

Involvement of peptidyl-prolyl isomerase Pin1 in the inhibitory effect of fluvastatin on endothelin-1-induced cardiomyocyte hypertrophy

Aims

Cardiac hypertrophy is elicited by endothelin (ET)-1 as well as other neurohumoral factors, hemodynamic overload, and oxidative stress; HMG-CoA reductase inhibitors (statins) were shown to inhibit cardiac hypertrophy partly via the anti-oxidative stress. One of their common intracellular pathways is the phosphorylation cascade of MEK signaling. Pin1 specifically isomerizes the phosphorylated protein with Ser/Thr-Pro bonds and regulates their activity through conformational changes. There is no report whether the Pin1 activation contributes to ET-1-induced cardiomyocyte hypertrophy and whether the Pin1 inactivation contributes to the inhibitory effect of statins. The aim of this study was to reveal these questions.

Main methods

We assessed neonatal rat cardiomyocyte hypertrophy using ET-1 and fluvastatin by the cell surface area, ANP mRNA expression, JNK and c-Jun phosphorylation, and [³H]-leucine incorporation.

Key findings

Fluvastatin inhibited ET-1-induced increase in the cell surface area, ANP expression, and [³H]-leucine incorporation; and it suppressed the signaling cascade from JNK to c-Jun. The phosphorylated Pin1 level, an inactive form, was decreased by ET-1; however, it reached basal level by fluvastatin. Furthermore, Pin1 overexpression clearly elicited cardiomyocyte hypertrophy, which was inhibited by fluvastatin.

Significance

This is the first report that ET-1-induced cardiomyocyte hypertrophy is mediated through the Pin1 activation and that the inhibitory effect of fluvastatin on cardiomyocyte hypertrophy would partly be attributed to the suppression of the Pin1 function. This study firstly suggests that Pin1 determines the size of hypertrophied cardiomyocyte by regulating the activity of phosphorylated molecules and that statins exert their pleiotropic effects partly via Pin1 inactivation.     

Pin1 acts catalytically to promote a conformational change in Cdc25

Pin1 is an essential protein that can peptidyl-prolyl-isomerize small phosphopeptides. It has been suggested that Pin1 regulates entry into mitosis by catalyzing the cis/trans-isomerization of prolines on critical protein substrates in response to phosphorylation. We show that Pin1 catalytically generates a conformational change on the mitotic phosphatase Cdc25, as assayed by limited protease digestion, differential reactivity to a phosphoserine-proline-directed monoclonal antibody (MPM-2), and by changes in Cdc25 enzymatic activity. Pin1 catalytically modifies the conformation of Cdc25 at stoichiometries less than 0.0005, and mutants of Pin1 in the prolyl isomerase domain are not active. We suggest that, although difficult to detect, phosphorylation-dependent conformational changes mediated by prolyl isomerization may play an important regulatory role in the cell cycle.     

Nerve growth factor stimulates interaction of Cayman ataxia protein BNIP-H/Caytaxin with peptidyl-prolyl isomerase Pin1 in differentiating neurons

Mutations in ATCAY that encodes the brain-specific protein BNIP-H (or Caytaxin) lead to Cayman cerebellar ataxia. BNIP-H binds to glutaminase, a neurotransmitter-producing enzyme, and affects its activity and intracellular localization. Here we describe the identification and characterization of the binding between BNIP-H and Pin1, a peptidyl-prolyl cis/trans isomerase. BNIP-H interacted with Pin1 after nerve growth factor-stimulation and they co-localized in the neurites and cytosol of differentiating pheochromocytoma PC12 cells and the embryonic carcinoma P19 cells. Deletional mutagenesis revealed two cryptic binding sites within the C-terminus of BNIP-H such that single point mutants affecting the WW domain of Pin1 completely abolished their binding. Although these two sites do not contain any of the canonical Pin1-binding motifs they showed differential binding profiles to Pin1 WW domain mutants S16E, S16A and W34A, and the catalytically inert C113A of its isomerase domain. Furthermore, their direct interaction would occur only upon disrupting the ability of BNIP-H to form an intramolecular interaction by two similar regions. Furthermore, expression of Pin1 disrupted the BNIP-H/glutaminase complex formation in PC12 cells under nerve growth factor-stimulation. These results indicate that nerve growth factor may stimulate the interaction of BNIP-H with Pin1 by releasing its intramolecular inhibition. Such a mechanism could provide a post-translational regulation on the cellular activity of BNIP-H during neuronal differentiation.     

The Ded1 DEAD box helicase interacts with Chk1 and Cdc2.

Ded1 is a fission yeast DEAD box protein involved in translation. We isolated Ded1 in a screen for multi-copy suppressors of a cold-sensitive, loss-of-function mutant of the cyclin-dependent kinase Cdc2. The checkpoint protein kinase Chk1, required for cell cycle arrest in response to DNA damage, was also isolated in this screen. Ded1 interacts with Chk1 in a two-hybrid screen, and this physical interaction can be recapitulated in Schizosaccharomyces pombe. The Ded1 polypeptide is modified in response to heat shock and depletion of carbon source. These two stressors appear to cause different modifications. Thus, the Ded1 protein appears to respond to particular types of cellular stress and may influence the activity of Cdc2 as a result.     

An irreversible inhibitor of peptidyl-prolyl cis/trans isomerase Pin1 and evaluation of cytotoxicity

Pin1 (protein interacting with never in mitosis A-1) is a member of the peptidyl prolyl isomerase (PPIase) family, and catalyzes cis-trans isomerization of pThr/Ser-Pro amide bonds. Because Pin1 is overexpressed in various cancer cell lines and promotes cell growth, it is considered a target for anticancer agents. Here, we designed and synthesized a covalently binding Pin1 inhibitor (S)-2 to target Pin1’s active site. This compound inhibited Pin1 in protease-coupled assay, and formed a covalent bond with Cys113 of Pin1, as determined by ESI-MS. The acetoxymethyl ester of (S)-2, i.e., 6, suppressed cyclin D1 expression in human prostate cancer PC-3 cells, and exhibited cytotoxicity. Pin1-knockdown experiments indicated that a target for the cytotoxicity of 6 is Pin1.     

The Polo-like kinase Plx1 interacts with and inhibits Myt1 after fertilization of Xenopus eggs

During the meiotic cell cycle in Xenopus oocytes, p90(rsk), the downstream kinase of the Mos-MAPK pathway, interacts with and inhibits the Cdc2 inhibitory kinase Myt1. However, p90(rsk) is inactivated after fertilization due to the degradation of Mos. Here we show that the Polo-like kinase Plx1, instead of p90(rsk), interacts with and inhibits Myt1 after fertilization of Xenopus eggs. At the M phase of the embryonic cell cycle, Cdc2 phosphorylates Myt1 on Thr478 and thereby creates a docking site for Plx1. Plx1 can phosphorylate Myt1 and inhibit its kinase activity both in vitro and in vivo. The interaction between Myt1 and Plx1 is required, at least in part, for normal embryonic cell divisions. Finally, and interestingly, Myt1 is phosphorylated on Thr478 even during the meiotic cell cycle, but its interaction with Plx1 is largely inhibited by p90(rsk)-mediated phosphorylation. These results indicate a switchover in the Myt1 inhibition mechanism at fertilization of Xenopus eggs, and strongly suggest that Plx1 acts as a direct inhibitory kinase of Myt1 in the mitotic cell cycles in Xenopus.     

Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex

We have discovered an early mitotic inhibitor, Emi1, which regulates mitosis by inhibiting the anaphase promoting complex/cyclosome (APC). Emi1 is a conserved F box protein containing a zinc binding region essential for APC inhibition. Emi1 accumulates before mitosis and is ubiquitylated and destroyed in mitosis, independent of the APC. Emi1 immunodepletion from cycling Xenopus extracts strongly delays cyclin B accumulation and mitotic entry, whereas nondestructible Emi1 stabilizes APC substrates and causes a mitotic block. Emi1 binds the APC activator Cdc20, and Cdc20 can rescue an Emi1-induced block to cyclin B destruction. Our results suggest that Emi1 regulates progression through early mitosis by preventing premature APC activation, and may help explain the well-known delay between cyclin B/Cdc2 activation and cyclin B destruction.     

Dual phosphorylation controls Cdc25 phosphatases and mitotic entry

Negative regulation of the Cdc25C protein phosphatase by phosphorylation on Ser 216, the 14-3-3-binding site, is an important regulatory mechanism used by cells to block mitotic entry under normal conditions and after DNA damage. During mitosis, Cdc25C is not phosphorylated on Ser 216 and ionizing radiation (IR) does not induce either phosphorylation of Ser 216, or binding to 14-3-3. Here, we show that Cdc25C is phosphorylated on Ser 214 during mitosis, which in turn prevents phosphorylation of Ser 216. Mutation of Ser 214 to Ala reconstitutes Ser 216 phosphorylation and 14-3-3 binding during mitosis. Introduction of exogenous Cdc25C(S214A) into HeLa cells depleted of endogenous Cdc25C results in a substantial delay to mitotic entry. This effect was fully reversed in a S214A/S216A double-mutant, implying that the inhibitory effect of S214A mutant was entirely dependent on Ser 216 phosphorylation. A similar regulatory mechanism may also apply to another mitotic phosphatase, Cdc25B, as well as mitotic phosphatases of other species, including Xenopus laevis. We propose that this pathway ensures that Cdc2 remains active once mitosis is initiated and is a key control mechanism for maintaining the proper order of cell-cycle transitions.