The lamin B receptor of the inner nuclear membrane undergoes mitosis-specific phosphorylation and is a substrate for p34cdc2-type protein kinase.

The lamin B receptor (LBR) is an integral protein of the inner nuclear membrane that interacts with lamin B in vitro. If contains a 204-amino acid nucleoplasmic amino-terminal domain and a hydrophobic carboxyl-terminal domain with eight putative transmembrane segments. We found cell cycle-dependent phosphorylation of LBR using phosphoamino acid analysis and phosphopeptide mapping of in vivo 32P-labeled LBR immunoprecipitated from chicken cells in interphase and arrested in mitosis. LBR was phosphorylated only on serine residues in interphase and on serine and threonine residues in mitosis. Some serine residues phosphorylated in interphase were not phosphorylated in mitosis. To identify a threonine residue specifically phosphorylated in mitosis and the responsible protein kinase, wild-type and mutant LBR nucleoplasmic domain fusion proteins were phosphorylated in vitro by p34cdc2-type protein kinase. Comparisons of phosphopeptide maps to those of in vivo 32P-labeled mitotic LBR showed that Thr188 is likely to be phosphorylated by this enzyme during mitosis. These phosphorylation/dephosphorylation events may be responsible for some of the changes in the interaction between the nuclear lamina and the inner nuclear membrane that occur during mitosis.     

Negative regulation of condensin I by CK2-mediated phosphorylation

Condensin I, which plays an essential role in mitotic chromosome assembly and segregation in vivo, constrains positive supercoils into DNA in the presence of adenosine triphosphate in vitro. Condensin I is constitutively present in a phosphorylated form throughout the HeLa cell cycle, but the sites at which it is phosphorylated in interphase cells differ from those recognized by Cdc2 during mitosis. Immunodepletion, in vitro phosphorylation, and immunoblot analysis using a phospho-specific antibody suggested that the CK2 kinase is likely to be responsible for phosphorylation of condensin I during interphase. In contrast to the slight stimulatory effect of Cdc2-induced phosphorylation of condensin I on supercoiling, phosphorylation by CK2 reduced the supercoiling activity of condensin I. CK2-mediated phosphorylation of condensin I is spatially and temporally regulated in a manner different to that of Cdc2-mediated phosphorylation: CK2-dependent phosphorylation increases during interphase and decreases on chromosomes during mitosis. These findings are the first to demonstrate a negative regulatory mode for condensin I, a process that may influence chromatin structure during interphase and mitosis.     

Cdc28-dependent regulation of the Cdc5/Polo kinase

Polo kinase is activated as cells enter mitosis and plays a central role in coordinating diverse mitotic events, yet the mechanisms leading to activation of Polo kinase are poorly understood . Work in Xenopus meiotic cell cycles has suggested that Polo kinase functions in a pathway that helps trigger activation of Cdk1 . However, studies in other organisms have suggested that activation of Polo kinase is dependent upon Cdk1 and therefore occurs downstream of Cdk1 activation . In this study, we have investigated the role of Cdk1 in the activation of budding yeast Polo kinase. The budding yeast homologs of Cdk1 and Polo kinase are referred to as Cdc28 and Cdc5. We show that signaling from Cdc28 is required to maintain Cdc5 activity in vivo. Furthermore, purified Cdc28 associated with the mitotic cyclin Clb2 is sufficient to activate purified Cdc5 in vitro. A single Cdc28 consensus phosphorylation site found at threonine 242 in the activation loop segment of Cdc5 is required for Cdc5 function in vivo and for kinase activity in vitro, whereas four other Cdc28 consensus sites are dispensable. Analysis of Cdc5 phosphorylation by mass spectrometry indicates that threonine 242 is phosphorylated in vivo. These results suggest that Cdc28 activates Cdc5 via phosphorylation of threonine 242.     

Identification of major nucleolar proteins as candidate mitotic substrates of cdc2 kinase

Following the identification of the cdc2 kinase as a major element controlling entry of cells into mitosis, it is important to define the physiological target range of this enzyme. Here, we demonstrate that two major nucleolar proteins, nucleolin and NO38, are highly phosphorylated during mitosis. Importantly, the two nucleolar proteins are also phosphorylated by highly purified starfish cdc2 kinase in vitro, on sites that correspond to those observed specifically during mitosis in vivo. A repeated motif (TPXKK) is identified as the likely mitotic phosphoacceptor site in nucleolin, in that a synthetic peptide mimicking this site functions as both a substrate and a competitive inhibitor of cdc2 kinase. These results identify two novel candidate substrates for cdc2 kinase, and they implicate protein phosphorylation in controlling mitotic changes in nucleolar structure and activity.     

Mitosis-specific phosphorylation of gar2, a fission yeast nucleolar protein structurally related to nucleolin

The nucleolar protein gar2 of fission yeast is structurally related to the multifunctional nucleolar protein nucleolin from vertebrates and has been shown to be implicated in production of 18S rRNA. gar2 contains several potential casein kinase 2 (CK2) phosphorylation sites and a single putative p34^cdc2 phosphorylation site in the consensus S₅₀PKK. Here, we show that, like nucleolin, gar2 is phosphorylated in vitro by both highly purified CK2 from CHO cells and p34^cdc2 from starfish oocytes. Moreover, the substitution of alanine for the N-terminal serine 50 abolishes phosphorylation by p34^cdc2 in vitro. We also provide evidence that gar2 is phosphorylated in vitro by a p13^suc1-Sepharose-bound kinase from Schizosaccharomyces pombe extracts that displays cell cycle-regulated activity similar to that of the p34^cdc2 kinase. In vivo ³²P labeling of cells indicates that gar2 is a phosphoprotein and that incorporation of phosphate on residue 50 occurs specifically at mitosis. Taken together, these results lead us to propose that gar2 is likely to be an in vivo substrate for the mitotic p34^cdc2 kinase. However, this posttranslational modification of the gar2 protein does not appear to be essential for normal production of 18S rRNA. Received: 5 September 1996; in revised form: 4 February 1997 / Accepted: 24 February 1997     

Analysis of the role of Aurora B on the chromosomal targeting of condensin I

During mitosis, chromosome condensation takes place, which entails the conversion of interphase chromatin into compacted mitotic chromosomes. Condensin I is a five-subunit protein complex that plays a central role in this process. Condensin I is targeted to chromosomes in a mitosis-specific manner, which is regulated by phosphorylation by mitotic kinases. Phosphorylation of histone H3at serine 10 (Ser10) occurs during mitosis and its physiological role is a longstanding question. We examined the function of Aurora B, a kinase that phosphorylates Ser10, in the chromosomal binding of condensin I and mitotic chromosome condensation, using an in vitro system derived from Xenopus egg extract. Aurora B depletion from a mitotic egg extract resulted in the loss of H3 phosphorylation, accompanied with a 50% reduction of chromosomal targeting of condensin I. Alternatively, a portion of condensin I was bound to sperm chromatin, and chromosome-like structures were assembled when okadaic acid (OA) was supplemented in an interphase extract that lacks Cdc2 activity. However, chromosomal targeting of condensin I was abolished when Aurora B was depleted from the OA-treated interphase extract. From these results, it is suggested that Aurora B-dependent and Cdc2-independent pathways of the chromosomal targeting of condensin I are present.     

Phosphorylation and activation of the Xenopus Cdc25 phosphatase in the absence of Cdc2 and Cdk2 kinase activity.

The M-phase inducer, Cdc25C, is a dual-specificity phosphatase that directly phosphorylates and activates the cyclin B/Cdc2 kinase complex, leading to initiation of mitosis. Cdc25 itself is activated at the G2/M transition by phosphorylation on serine and threonine residues. Previously, it was demonstrated that Cdc2 kinase is capable of phosphorylating and activating Cdc25, suggesting the existence of a positive feedback loop. In the present study, kinases other than Cdc2 that can phosphorylate and activate Cdc25 were investigated. Cdc25 was found to be phosphorylated and activated by cyclin A/Cdk2 and cyclin E/Cdk2 in vitro. However, in interphase Xenopus egg extracts with no detectable Cdc2 and Cdk2, treatment with the phosphatase inhibitor microcystin activated a distinct kinase that could phosphorylate and activate Cdc25. Microcystin also induced other mitotic phenomena such as chromosome condensation and nuclear envelope breakdown in extracts containing less than 5% of the mitotic level of Cdc2 kinase activity. These findings implicate a kinase other than Cdc2 and Cdk2 that may initially activate Cdc25 in vivo and suggest that this kinase may also phosphorylate M-phase substrates even in the absence of Cdc2 kinase.     

Cell cycle regulation of a Xenopus Wee1-like kinase.

Using a polymerase chain reaction-based strategy, we have isolated a gene encoding a Wee1-like kinase from Xenopus eggs. The recombinant Xenopus Wee1 protein efficiently phosphorylates Cdc2 exclusively on Tyr-15 in a cyclin-dependent manner. The addition of exogenous Wee1 protein to Xenopus cell cycle extracts results in a dose-dependent delay of mitotic initiation that is accompanied by enhanced tyrosine phosphorylation of Cdc2. The activity of the Wee1 protein is highly regulated during the cell cycle: the interphase, underphosphorylated form of Wee1 (68 kDa) phosphorylates Cdc2 very efficiently, whereas the mitotic, hyperphosphorylated version (75 kDa) is weakly active as a Cdc2-specific tyrosine kinase. The down-modulation of Wee1 at mitosis is directly attributable to phosphorylation, since dephosphorylation with protein phosphatase 2A restores its kinase activity. During interphase, the activity of this Wee1 homolog does not vary in response to the presence of unreplicated DNA. The mitosis-specific phosphorylation of Wee1 is due to at least two distinct kinases: the Cdc2 protein and another activity (kinase X) that may correspond to an MPM-2 epitope kinase. These studies indicate that the down-regulation of Wee1-like kinase activity at mitosis is a multistep process that occurs after other biochemical reactions have signaled the successful completion of S phase.     

Nif1, a novel mitotic inhibitor in Schizosaccharomyces pombe.

In Schizosaccharomyces pombe, the activity of the M-phase-inducing Cdc2/Cdc13 cyclin-dependent kinase is inhibited by Wee1 and Mik1 tyrosine kinases, and activated by Cdc25 and Pyp3 tyrosine phosphatases. Cdc2/Cdc13 activity is also indirectly regulated by the approximately 70 kDa Nim1 (Cdrl) serine/threonine kinase, which promotes mitosis by inhibiting Wee1 via direct phosphorylation. To understand better the function and regulation of Nim1, the yeast two-hybrid system was used to isolate S.pombe cDNA clones encoding proteins that interact with Nim1. Sixteen of the 17 cDNA clones were derived from the same gene, named nif1 + (nim1 interacting factor-1). Nif1 is a novel approximately 75 kDa protein containing a leucine zipper motif. The Nif1-Nim1 interaction requires a small region of Nim1 that immediately follows the N-terminal catalytic domain. This region is required for Nim1 activity both in vivo and in vitro. delta nif1 mutants are approximately 10% smaller than wild type, indicating that Nif1 is involved in inhibiting the onset of mitosis. Consistent with this proposal, overproduction of Nif1 was found to cause a cell elongation phenotype that is very similar to delta nim1 mutants. Nif1 overproduction causes cell cycle arrest in cells that are partly defective for Cdc25 activity, but has no effect in delta nim1 or delta wee1 mutants. Nif1 also inhibits Nim1-mediated phosphorylation of Wee1 in an insect cell expression system. These observations strongly suggest that Nif1 negatively regulates the onset of mitosis by a novel mechanism, namely inhibiting Nim1 kinase.     

Phosphorylation of threonine 10 on CKBBP1/SAG/ROC2/Rbx2 by protein kinase CKII promotes the degradation of IkappaBalpha and p27Kip1

In eukaryotic cells, protein kinase CKII is required for progression through the cell division cycle. We recently reported that CKBBP1/SAG/ROC2/Rbx2 associates with the beta-subunit of CKII and is phosphorylated by purified CKII in the presence of ATP in vitro. In this report, we demonstrate that CKBBP1 is efficiently phosphorylated in vitro by purified CKII in the presence of GTP and by heparin-sensitive protein kinase in HeLa cell extract. Mutational analysis indicates that CKII phosphorylates threonine at residue 10 within CKBBP1. Furthermore, CKBBP1 is phosphorylated in vivo and threonine to alanine mutation at residue 10 abrogates the phosphorylation of CKBBP1 observed in vivo, indicating that CKII is a major kinase that is responsible for in vivo phosphorylation of CKBBP1. As compared with the wild-type CKBBP1 or CKBBP1T10E (in which threonine 10 is replaced by glutamate), overexpression of nonphosphorylatable CKBBP1 (CKBBP1T10A) results in accumulation of IkappaBalpha and p27Kip1. Experiments using proteasome inhibitor MG132 and CKII inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole suggest that the accumulation of IkappaBalpha and p27Kip1 results primarily from the reduction of proteasomal degradation in cells expressing CKBBP1T10A, and that CKII-mediated CKBBP1 phosphorylation is required for efficient degradation of IkappaBalpha and p27Kip1. Overexpression of CKBBP1T10A in HeLa cells suppresses cell proliferation and causes accumulation of G1/G0 peak of the cell cycle. Taken together, our results indicate that CKII may control IkappaBalpha and p27Kip1 degradation and thereby G1/S phase transition through the phosphorylation of threonine 10 within CKBBP1.     

Distinct phosphoisoforms of the Xenopus Mcm4 protein regulate the function of the Mcm complex

Initiation of DNA replication in eukaryotes requires the assembly of prereplication complexes (pre-Rcs) at the origins of replication. The assembly and function of the pre-Rcs appear to be controlled by phosphorylation events. In this study we report the detailed characterization of the cell cycle phosphorylation of one component of the Xenopus pre-Rcs, the Mcm protein complex. We show that individual Mcm subunits are differentially phosphorylated during the cell cycle. During mitosis, the Mcm4 subunit is hyperphosphorylated, while the other subunits are not actively phosphorylated. The mitotic phosphorylation of Mcm4 requires Cdc2-cyclin B and other unknown kinases. Following exit from mitosis, the Mcm4 subunit of the cytosolic interphase complex undergoes dephosphorylation, and the Mcm2, Mcm3, or Mcm6 subunits are then actively phosphorylated by kinase(s) other than cyclin-dependent kinases (Cdks) or Cdc7. The association of the Mcm complex with the pre-Rcs correlates with the formation of a transient interphase complex. This complex contains an intermediately phosphorylated Mcm4 subunit and is produced by partial dephosphorylation of the mitotic hyperphosphorylated Mcm4 protein. Complete dephosphorylation of the Mcm4 subunit inactivates the Mcm complex and prevents its binding to the chromatin. Once the Mcm complex is assembled on the chromatin the Mcm4 and the Mcm2 proteins are the only subunits phosphorylated during the activation of the pre-Rcs. These chromatin-associated phosphorylations require nuclear transport and are independent of Cdk2-cyclin E. These results suggest that the changes in Mcm4 phosphorylation regulate pre-Rc assembly and the function of the pre-Rcs on the chromatin.     

Dynamic Changes in Nuclear Architecture during Mitosis: On the Role of Protein Phosphorylation in Spindle Assembly and Chromosome Segregation

During mitosis, the vertebrate cell nucleus undergoes profound changes in architecture. At the onset of mitosis, the nuclear envelope breaks down, the nuclear lamina is depolymerized, and interphase chromatin is condensed to chromosomes. Concomitantly, cytoplasmic microtubules are reorganized into a mitotic spindle apparatus, a highly dynamic structure required for the segregation of sister chromatids. Many of the above events are controlled by reversible phosphorylation. Hence, our laboratory is interested in characterizing the kinases involved in promoting progression through mitosis and in identifying their relevant substrates. Prominent among the kinases responsible for regulating entry into mitosis is the Cdc2 kinase, the first member of the cyclin dependent kinase (Cdk) family. Recently, we found that Cdc2 phosphorylates HsEg5, a human kinesin-related motor protein associated with centrosomes and the spindle apparatus. Our results indicate that phosphorylation regulates the association of HsEg5 with the mitotic spindle and that the function of this plus-end directed motor is essential for centrosome separation and bipolar spindle formation. Another kinase implicated in regulating progression through mitosis is Plk1 (polo-like kinase 1), the human homologue of theDrosophilagene product “polo.” By antibody microinjection we have found that Plk1 is required for the functional maturation of centrosomes and hence for entry into mitosis. Furthermore, we found that microinjected anti-Plk1 antibodies caused a more severe block to cell cycle progression in diploid fibroblasts than in immortalized tumor cells. This observation hints at the existence of a checkpoint linking Cdc2 activation to the presence of functional centrosomes.     

Phosphorylation of ribosomal protein L5 by protein kinase CKII decreases its 5S rRNA binding activity.

We have recently reported that ribosomal protein L5 associates with the beta subunit of protein kinase CKII (CKII) (Kim, J.-M., Cha, J. -Y., Marshak, D. R., and Bae, Y.-S. (1996) Biochem. Biophys. Res. Commun. 226, 180-186). In this study, we demonstrate that CKII is able to catalyze the phosphorylation of the human L5 protein in vitro, which results in a decrease in 5S rRNA binding activity. Phosphoamino acid analysis indicated that the phosphorylation occurs on serine residues. Sequence analysis of cyanogen bromide-digested phosphopeptides and analysis of L5 deletion mutants indicates that the main phosphorylated residues are located within two fragments corresponding of residues 142-200 and residues 272-297 of the human L5. Based on our present results, we suggest that the phosphorylation of L5 by CKII is one of the mechanisms that regulates nucleolar targeting of 5S rRNA and/or ribosome assembly in the cell.     

Cdc25 regulates the phosphorylation and activity of the Xenopus cdk2 protein kinase complex.

The Xenopus cdk2 gene encodes a 32-kDa protein kinase with sequence similarity to the 34-kDa product of the cdc2 gene. Previous studies have shown that the kinase activity of the protein product of the cdk2 gene oscillates in the Xenopus embryonic cell cycle with a high in M-phase and a low in interphase. In the present study cdk2 was found not to be associated with any newly synthesized proteins during the cell cycle, but the enzyme did undergo periodic changes in phosphorylation. Upon exit from metaphase, cdk2 became increasingly phosphorylated on both tyrosine and serine residues, and labeling on these residues increased progressively until entry into mitosis, when tyrosine residues were markedly dephosphorylated. Phosphopeptide mapping of cdk2 demonstrated the major sites of phosphorylation were in a phosphopeptide with a pI of 3.7 that contained both phosphoserine and phosphotyrosine. This phosphopeptide accumulated in egg extracts blocked in S-phase with aphidicolin and was not evident in cdc2 immunoprecipitated under the same conditions. Under the same conditions cdc2 was phosphorylated primarily on a phosphopeptide containing both phosphothreonine and phosphotyrosine residues, most likely threonine 14 and tyrosine 15. Affinity-purified human GST-cdc25 was able to dephosphorylate and activate cdk2 isolated from interphase cells. Phosphopeptide mapping demonstrated that the phosphate was specifically removed from the same phosphopeptide identified as the major in vivo site of phosphorylation. These results demonstrate that cdk2 is regulated in the cell cycle by phosphorylation and dephosphorylation on both serine and tyrosine residues. Moreover, the increased phosphorylation of cdk2 in aphidicolin-blocked extracts and the ability of cdc25 to mediate cdk2 dephosphorylation in vitro suggest the possibility that cdk2 is part of the mechanism ensuring mitosis is not initiated until completion of DNA replication. It also implies cdc25 may have other functions in addition to the regulation of cdc2 kinase activity.     

Cdc42-induced activation of the mixed-lineage kinase SPRK in vivo. Requirement of the Cdc42/Rac interactive binding motif and changes in phosphorylation

Src homology 3 domain (SH3)-containing proline-rich protein kinase (SPRK)/mixed-lineage kinase (MLK)-3 is a serine/threonine kinase that upon overexpression in mammalian cells activates the c-Jun NH(2)-terminal kinase pathway. The mechanisms by which SPRK activity is regulated are not well understood. The small Rho family GTPases, Rac and Cdc42, have been shown to bind and modulate the activities of signaling proteins, including SPRK, which contain Cdc42/Rac interactive binding motifs. Coexpression of SPRK and activated Cdc42 increases SPRKs activity. SPRKs Cdc42/Rac interactive binding-like motif contains six of the eight consensus residues. Using a site-directed mutagenesis approach, we show that SPRK contains a functional Cdc42/Rac interactive binding motif that is required for SPRKs association with and activation by Cdc42. However, experiments using a SPRK variant that lacks the COOH-terminal zipper region/basic stretch suggest that this region may also contribute to Cdc42 binding. Unlike the PAK family of protein kinases, we find that the activation of SPRK by Cdc42 cannot be recapitulated in an in vitro system using purified, recombinant proteins. Comparative phosphopeptide mapping demonstrates that coexpression of activated Cdc42 with SPRK alters the in vivo serine/threonine phosphorylation pattern of SPRK suggesting that the mechanism by which Cdc42 increases SPRKs catalytic activity involves a change in the in vivo phosphorylation of SPRK. This is, to the best of our knowledge, the first demonstrated example of a Cdc42-mediated change in the in vivo phosphorylation of a protein kinase. These studies suggest an additional component or cellular environment is required for SPRK activation by Cdc42.     

A human homolog of Drosophila warts tumor suppressor, h-warts, localized to mitotic apparatus and specifically phosphorylated during mitosis.

We identified a human homolog of Drosophila warts tumor suppressor gene, termed h-warts, which was mapped at chromosome 6q24-25.1. The h-warts protein has a serine/threonine kinase domain and is localized to centrosomes in interphase cells. However, it becomes localized to the mitotic apparatus, including spindle pole bodies, mitotic spindle, and midbody, in a highly dynamic manner during mitosis. Furthermore, h-warts is specifically phosphorylated in cells at mitotic phase, most likely by Cdc2 kinase. These findings suggest that h-warts functions as a component of the mitotic apparatus and is involved in proper progression of mitosis.     

A positive feedback loop between protein kinase CKII and Cdc37 promotes the activity of multiple protein kinases

We report here the identification of CDC37, which encodes a putative Hsp90 co-chaperone, as a multicopy suppressor of a temperature-sensitive allele (cka2-13(ts)) of the CKA2 gene encoding the alpha' catalytic subunit of protein kinase CKII. Unlike wild-type cells, cka2-13 cells were sensitive to the Hsp90-specific inhibitor geldanamycin, and this sensitivity was suppressed by overexpression of either Hsp90 or Cdc37. However, only CDC37 was capable of suppressing the temperature sensitivity of a cka2-13 strain, implying that Cdc37 is the limiting component. Immunoprecipitation of metabolically labeled Cdc37 from wild-type versus cka2-13 strains revealed that Cdc37 is a physiological substrate of CKII, and Ser-14 and/or Ser-17 were identified as the most likely sites of CKII phosphorylation in vivo. A cdc37-S14,17A strain lacking these phosphorylation sites exhibited severe growth and morphological defects that were partially reversed in a cdc37-S14,17E strain. Reduced CKII activity was observed in both cdc37-S14A and cdc37-S17A mutants at 37 degrees C, and cdc37-S14A or cdc37-S14,17A overexpression was incapable of protecting cka2-13 mutants on media containing geldanamycin. Additionally, CKII activity was elevated in cells arrested at the G(1) and G(2)/M phases of the cell cycle, the same phases during which Cdc37 function is essential. Collectively, these data define a positive feedback loop between CKII and Cdc37. Additional genetic assays demonstrate that this CKII/Cdc37 interaction positively regulates the activity of multiple protein kinases in addition to CKII.     

Expression and secretion of human apolipoprotein A-I in the heart

Nuclear envelope-peripheral heterochromatin fractions contain multiple histone kinase activities. In vitro assays and amino-terminal sequencing show that one of these activities co-isolates with heterochromatin protein 1 (HP1) and phosphorylates histone H3 at threonine 3. Antibodies recognizing this post-translational modification reveal that in vivo phosphorylation at threonine 3 commences at early prophase in the vicinity of the nuclear envelope, spreads to pericentromeric chromatin during prometaphase and is fully reversed by late anaphase. This spatio-temporal pattern is distinct from H3 phosphorylation at serine 10, which also occurs during cell division, suggesting segregation of differentially phosphorylated chromatin to different regions of mitotic chromosomes.