Biomedical subjects

Philipp Kaldis

Publications and source records attributed to Philipp Kaldis.

At least 19 recordsLinked to original sources

PubMed2007

Kinase-independent function of cyclin E.

E-type cyclins are thought to drive cell-cycle progression by activating cyclin-dependent kinases, primarily CDK2. We previously found that cyclin E-null cells failed to incorporate MCM helicase into DNA prereplication complex during G(0) --> S phase progression. We now report that a kinase-deficient cyclin E mutant can partially restore MCM loading and S phase entry in cyclin E-null cells. We found that cyclin E is loaded onto chromatin during G(0) --> S progression. In the absence of cyclin E, CDT1 is normally loaded onto chromatin, whereas MCM is not, indicating that cyclin E acts between CDT1 and MCM loading. We observed a physical association of cyclin E with CDT1 and with MCMs. We propose that cyclin E facilitates MCM loading in a kinase-independent fashion, through physical interaction with CDT1 and MCM. Our work indicates that-in addition to their function as CDK activators-E cyclins play kinase-independent functions in cell-cycle progression.

Animals↗

PubMed2006

PRKAR1A inactivation leads to increased proliferation and decreased apoptosis in human B lymphocytes.

The multiple neoplasia syndrome Carney complex (CNC) is caused by heterozygote mutations in the gene, which codes for the RIalpha regulatory subunit (PRKAR1A) of protein kinase A. Inactivation of PRKAR1A and the additional loss of the normal allele lead to tumors in CNC patients and increased cyclic AMP signaling in their cells, but the oncogenetic mechanisms in affected tissues remain unknown. Previous studies suggested that PRKAR1A down-regulation may lead to increased mitogen-activated protein kinase (MAPK) signaling. Here, we show that, in lymphocytes with PRKAR1A-inactivating mutations, there is increased extracellular signal-regulated kinase (ERK) 1/2 and B-raf phosphorylation and MAPK/ERK kinase 1/2 and c-Myc activation, whereas c-Raf-1 is inhibited. These changes are accompanied by increased cell cycle rates and decreased apoptosis that result in an overall net gain in proliferation and survival. In conclusion, inactivation of PRKAR1A leads to widespread changes in molecular pathways that control cell cycle and apoptosis. This is the first study to show that human cells with partially inactivated RIalpha levels have increased proliferation and survival, suggesting that loss of the normal allele in these cells is not necessary for these changes to occur.

Apoptosis↗

PubMed2006

Identification of yin-yang regulators and a phosphorylation consensus for male germ cell-associated kinase (MAK)-related kinase.

MAK (male germ cell-associated protein kinase) and MRK/ICK (MAK-related kinase/intestinal cell kinase) are human homologs of Ime2p in Saccharomyces cerevisiae and of Mde3 and Pit1 in Schizosaccharomyces pombe and are similar to human cyclin-dependent kinase 2 (CDK2) and extracellular signal-regulated kinase 2 (ERK2). MAK and MRK require dual phosphorylation in a TDY motif catalyzed by an unidentified human threonine kinase and tyrosine autophosphorylation. Herein, we establish that human CDK-related kinase CCRK (cell cycle-related kinase) is an activating T157 kinase for MRK, whereas active CDK7/cyclin H/MAT1 complexes phosphorylate CDK2 but not MRK. Protein phosphatase 5 (PP5) interacts with MRK in a complex and dephosphorylates MRK at T157 in vitro and in situ. Thus, CCRK and PP5 are yin-yang regulators of T157 phosphorylation. To determine a substrate consensus, we screened a combinatorial peptide library with active MRK. MRK preferentially phosphorylates R-P-X-S/T-P sites, with the preference for arginine at position -3 (P-3) being more stringent than for prolines at P-2 and P+1. Using the consensus, we identified a putative phosphorylation site (RPLT(1080)S) for MRK in human Scythe, an antiapoptotic protein that interacts with MRK. MRK phosphorylates Scythe at T1080 in vitro as determined by site-directed mutagenesis and mass spectrometry, supporting the consensus and suggesting Scythe as a physiological substrate for MRK.

Amino Acid Motifs↗

PubMed2006

Dependence of cisplatin-induced cell death in vitro and in vivo on cyclin-dependent kinase 2.

Cisplatin is one of the most effective chemotherapeutics, but its usefulness is limited by its toxicity to normal tissues, including cells of the kidney proximal tubule. The purpose of these studies was to determine the mechanism of cisplatin cytotoxicity. It was shown in vivo that cisplatin administration induces upregulation of the gene for the p21 cyclin-dependent kinase (cdk) inhibitor in kidney cells. This protein is a positive effector on the fate of cisplatin-exposed renal tubule cells in vivo and in vitro; adenoviral transduction of p21 completely protected proximal tubule cells from cisplatin toxicity. Herein is reported that cdk2 inhibitory drugs protect kidney cells in vivo and in vitro, that transduction of kidney cells in vitro with dominant-negative cdk2 also protected, and that cdk2 knockout cells were resistant to cisplatin. The cdk2 knockout cells regained cisplatin sensitivity after transduction with wild-type cdk2. It is concluded that cisplatin cytotoxicity depends on cdk2 activation and that the mechanism of p21 protection is by direct inhibition of cdk2. This demonstrated the involvement of a protein that previously was associated with cell-cycle progression with pathways of apoptosis. It also was demonstrated that this pathway of cisplatin-induced cell death can be interceded in vivo to prevent nephrotoxicity.

Adenine↗

PubMed2006

CDK2 is required by MYC to induce apoptosis.

Depending upon the cellular and physiologic context, the overexpression of the MYC proto-oncogene results in rapid cell growth, proliferation, induction of apoptosis and/or proliferative arrest. What determines the precise consequences upon MYC activation is not clear. We have found that cyclin-dependent kinase 2 (CDK2) is required by MYC to induce apoptosis. MYC-induced apoptosis was suppressed in mouse embryonic fibroblasts (MEF) knocked out for Cdk2 or normal human fibroblasts (NHF) upon expression of the CDK2 inhibitor p27 or treated with RNAi directed at CDK2. Knockout of Cdk2 did not prevent MYC from inducing p53 and Bim. The inhibition of CDK2 did not prevent apoptosis induced by the DNA damaging agent etoposide. Our results surprisingly suggest that CDK2 defines whether MYC induction causes apoptosis.

Animals↗

PubMed2006

Cdk2 and Cdk4 cooperatively control the expression of Cdc2.

Progression through the mammalian cell cycle is associated with the activity of four cyclin dependent kinases (Cdc2/Cdk1, Cdk2, Cdk4, and Cdk6). Knockout mouse models have provided insight into the interplay of these Cdks. Most of these models do not exhibit major cell cycle defects revealing redundancies, and suggesting that a single Cdk might be sufficient to drive the cell cycle, similar as in yeast. Recent work on Cdk2/Cdk4 double knockouts has indicated that these two Cdks are required to phosphorylate Rb during late embryogenesis. The lack of Rb phosphorylation is progressive and associated with reduced E2F-inducible gene expression. Cdk2 and Cdk4 share the essential function of coupling the G1/S transition with mitosis. However, proliferation in early embryogenesis appears to be independent of Cdk2 and Cdk4. We discuss these observations and propose molecular mechanisms that establish the requirement for Cdk2 and Cdk4 at the G1/S transition. We are considering that the balance between proliferation and differentiation is disturbed, which affects especially heart development and leads to embryonic lethality in Cdk2-/- Cdk4-/- mutants. We also discuss the specific functions of Cdk4 and Cdk6, which ironically do not compensate for each other.

Journal Article↗

PubMed2006

Cell Division, a new open access online forum for and from the cell cycle community.

Cell Division is a new, open access, peer-reviewed online journal that publishes cutting-edge articles, commentaries and reviews on all exciting aspects of cell cycle control in eukaryotes. A major goal of this new journal is to publish timely and significant studies on the aberrations of the cell cycle network that occur in cancer and other diseases.

Journal Article↗

PubMed2006

IL-7 promotes T cell proliferation through destabilization of p27Kip1.

Interleukin (IL)-7 is required for survival and homeostatic proliferation of T lymphocytes. The survival effect of IL-7 is primarily through regulation of Bcl-2 family members; however, the proliferative mechanism is unclear. It has not been determined whether the IL-7 receptor actually delivers a proliferative signal or whether, by promoting survival, proliferation results from signals other than the IL-7 receptor. We show that in an IL-7-dependent T cell line, cells protected from apoptosis nevertheless underwent cell cycle arrest after IL-7 withdrawal. This arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27Kip1 through a posttranslational mechanism. Overexpression of p27Kip1 induced G1 arrest in the presence of IL-7, whereas knockdown of p27Kip1 by small interfering RNA promoted S phase entry after IL-7 withdrawal. CD4 or CD8 T cells transferred into IL-7-deficient hosts underwent G1 arrest, whereas 27Kip1-deficient T cells underwent proliferation. We observed that IL-7 withdrawal activated protein kinase C (PKC)theta and that inhibition of PKCtheta with a pharmacological inhibitor completely blocked the rise of p27Kip1 and rescued cells from G1 arrest. The conventional pathway to breakdown of p27Kip1 is mediated by S phase kinase-associated protein 2; however, our evidence suggests that PKCtheta acts via a distinct, unknown pathway inducing G1 arrest after IL-7 withdrawal from T cells. Hence, IL-7 maintains T cell proliferation through a novel pathway of p27Kip1 regulation.

Animals↗

PubMed2006

p205, a potential tumor suppressor, inhibits cell proliferation via multiple pathways of cell cycle regulation.

p205 is a member of the interferon-inducible p200 family of proteins that regulate cell proliferation. Over-expression of p205 inhibits cell growth, although its mechanism of action is currently unknown. Therefore, we evaluated the effect of p205 on the p53 and Rb-dependent pathways of cell cycle regulation. p205 expression results in elevated levels of p21, and activates the p21 promoter in vitro in a p53-dependent manner. In addition, p205 induces increased expression of Rb, and binds directly to Rb and p53. Interestingly, p205 also induces growth inhibition independent of p53 and Rb by delaying G2/M progression in proliferating cells, and is a substrate for Cdk2 kinase activity. Finally, we have identified other binding partners of p205 by a yeast two-hybrid screen, including the paired homeodomain protein HoxB2. Taken together, our results indicate that p205 induces growth arrest by interaction with multiple transcription factors that regulate the cell cycle, including but not entirely dependent on the Rb- and p53-mediated pathways of growth inhibition.

Animals↗

PubMed2006

Mouse models of cell cycle regulators: new paradigms.

In yeast, a single cyclin-dependent kinase (Cdk) is able to regulate diverse cell cycle transitions (S and M phases) by associating with multiple stage-specific cyclins. The evolution of multicellular organisms brought additional layers of cell cycle regulation in the form of numerous Cdks, cyclins and Cdk inhibitors to reflect the higher levels of organismal complexity. Our current knowledge about the mammalian cell cycle emerged from early experiments using human and rodent cell lines, from which we built the current textbook model of cell cycle regulation. In this model, the functions of different cyclin/Cdk complexes were thought to be specific for each cell cycle phase. In the last decade, studies using genetically engineered mice in which cell cycle regulators were targeted revealed many surprises. We discovered the in vivo functions of cell cycle proteins within the context of a living animal and whether they are essential for animal development. In this review, we discuss first the textbook model of cell cycle regulation, followed by a global overview of data obtained from different mouse models. We describe the similarities and differences between the phenotypes of different mouse models including embryonic lethality, sterility, hematopoietic, pancreatic, and placental defects. We also describe the role of key cell cycle regulators in the development of tumors in mice, and the implications of these data for human cancer. Furthermore, animal models in which two or more genes are ablated revealed which cell cycle regulators interact genetically and functionally complement each other. We discuss for example the interaction of cyclin D1 and p27 and the compensation of Cdk2 by Cdc2. We also focus on new functions discovered for certain cell cycle regulators such as the regulation of S phase by Cdc2 and the role of p27 in regulating cell migration. Finally, we conclude the chapter by discussing the limitations of animal models and to what extent can the recent findings be reconciled with the past work to come up with a new model for cell cycle regulation with high levels of redundancy among the molecular players.

Animals↗

PubMed2006

Combined loss of Cdk2 and Cdk4 results in embryonic lethality and Rb hypophosphorylation.

Mouse knockouts of Cdk2 and Cdk4 have demonstrated that, individually, these genes are not essential for viability. To investigate whether there is functional redundancy, we have generated double knockout (DKO) mice. Cdk2-/- Cdk4-/- DKOs die during embryogenesis around E15 as a result of heart defects. We observed a gradual decrease of Retinoblastoma protein (Rb) phosphorylation and reduced expression of E2F-target genes, like Cdc2 and cyclin A2, during embryogenesis and in embryonic fibroblasts (MEFs). DKO MEFs are characterized by a decreased proliferation rate, impaired S phase entry, and premature senescence. HPV-E7-mediated inactivation of Rb restored normal expression of E2F-inducible genes, senescence, and proliferation in DKO MEFs. In contrast, loss of p27 did not rescue Cdk2-/- Cdk4-/- phenotypes. Our results demonstrate that Cdk2 and Cdk4 cooperate to phosphorylate Rb in vivo and to couple the G1/S phase transition to mitosis via E2F-dependent regulation of gene expression.

Animals↗

PubMed2005

Cell cycle sibling rivalry: Cdc2 vs. Cdk2.

It has been long believed that the cyclin-dependent kinase 2 (Cdk2) binds to cyclin E or cyclin A and exclusively promotes the G1/S phase transition and that Cdc2/cyclin B complexes play a major role in mitosis. We now provide evidence that Cdc2 binds to cyclin E (in addition to cyclin A and B) and is able to promote the G1/S transition. This new concept indicates that both Cdk2 and/or Cdc2 can drive cells through G1/S phase in parallel. In this review we discuss the classic cell cycle model and how results from knockout mice provide new evidence that refute this model. We focus on the roles of Cdc2 and p27 in regulating the mammalian cell cycle and propose a new model for cell cycle regulation that accommodates these novel findings.

Animals↗

PubMed2005

Biochemical characterization of Cdk2-Speedy/Ringo A2.

BACKGROUND: Normal cell cycle progression requires the precise activation and inactivation of cyclin-dependent protein kinases (CDKs), which consist of a CDK and a cyclin subunit. A novel cell cycle regulator called Speedy/Ringo shows no sequence similarity to cyclins, yet can directly bind to and activate CDKs. Speedy/Ringo proteins, which bind to and activate Cdc2 and Cdk2 in vitro, are required for the G2 to M transition during Xenopus oocyte maturation and for normal S-phase entry in cultured human cells. RESULTS: We have characterized the substrate specificity and enzymatic activity of human Cdk2-Speedy/Ringo A2 in order to gain insights into the possible functions of this complex. In contrast to Cdk2-cyclin A, which has a well-defined consensus target site ((S/T)PX(K/R)) that strongly favors substrates containing a lysine at the +3 position of substrates, Cdk2-Speedy/Ringo A2 displayed a broad substrate specificity at this position. Consequently, Cdk2-Ringo/Speedy A2 phosphorylated optimal Cdk2 substrates such as histone H1 and a KSPRK peptide poorly, only approximately 0.08% as well as Cdk2-cyclin A, but non-canonical Cdk2 substrates such as a KSPRY peptide relatively well, with an efficiency of approximately 80% compared to Cdk2-cyclin A. Cdk2-Speedy/Ringo A2 also phosphorylated authentic Cdk2 substrates, such as Cdc25 proteins, which contain non-canonical CDK phosphorylation sites, nearly as well as Cdk2-cyclin A. Phosphopeptide mapping indicated that Cdk2-Speedy/Ringo A2 and Cdk2-cyclin A phosphorylate distinct subsets of sites on Cdc25 proteins. Thus, the low activity that Cdk2-Speedy/Ringo A2 displays when assayed on conventional Cdk2 substrates may significantly underestimate the potential physiological importance of Cdk2-Speedy/Ringo A2 in phosphorylating key subsets of Cdk2 substrates. Unlike Cdk2-cyclin A, whose activity depends strongly on activating phosphorylation of Cdk2 on Thr-160, neither the overall catalytic activity nor the substrate recognition by Cdk2-Speedy/Ringo A2 was significantly affected by this phosphorylation. Furthermore, Cdk2-Speedy/Ringo A2 was not a suitable substrate for metazoan CAK (which phosphorylates Cdk2 at Thr-160), supporting the notion that Speedy/Ringo A2 activates Cdk2 in a CAK-independent manner. CONCLUSION: There are major differences in substrate preferences between CDK-Speedy/Ringo A2 and Cdk2-cyclin complexes. These differences may accommodate the CAK-independent activation of Cdk2 by Speedy/Ringo A2 and they raise the possibility that CDK-Speedy/Ringo A2 complexes could phosphorylate and regulate a subset of non-canonical CDK substrates, such as Cdc25 protein phosphatases, to control cell cycle progression.

Amino Acid Substitution↗

PubMed2005

Cdc2-cyclin E complexes regulate the G1/S phase transition.

The cyclin-dependent kinase inhibitor p27(Kip1) is known as a negative regulator of cell-cycle progression and as a tumour suppressor. Cdk2 is the main target of p27 (refs 2, 3) and therefore we hypothesized that loss of Cdk2 activity should modify the p27(-/-) mouse phenotype. Here, we show that although p27(-/-) Cdk2(-/-) mice developed ovary tumours and tumours in the anterior lobe of the pituitary, we failed to detect any functional complementation in p27(-/-) Cdk2(-/-) double-knockout mice, indicating a parallel pathway regulated by p27. We observed elevated levels of S phase and mitosis in tissues of p27(-/-) Cdk2(-/-) mice concomitantly with elevated Cdc2 activity in p27(-/-) Cdk2(-/-) extracts. p27 binds to Cdc2, cyclin B1, cyclin A2, or suc1 complexes in wild-type and Cdk2(-/-) extracts. In addition, cyclin E binds to and activates Cdc2. Our in vivo results provide strong evidence that Cdc2 may compensate the loss of Cdk2 function.

2-Aminopurine↗

PubMed2005

The N-terminal peptide of the Kaposi's sarcoma-associated herpesvirus (KSHV)-cyclin determines substrate specificity.

Cyclin-dependent kinases (Cdks) are activated by cyclin binding and phosphorylation by the Cdk-activating kinase (CAK). Activation of Cdk6 by the D-type cyclins requires phosphorylation of Cdk6 by CAK on threonine 177. In contrast, Cdk6 is activated by the Kaposi's sarcoma-associated herpesvirus (KSHV)-cyclin in the absence and presence of CAK phosphorylation. The activity of Cdk6.KSHV-cyclin complexes was investigated here by analyzing mutants of the KSHV-cyclin and Cdk6 in vitro as well as in U2OS cells. Deletion of the N terminus of the KSHV-cyclin affects the substrate specificity indicating that the N terminus is required for phosphorylation of histone H1 but not for other substrates. Mutation of residues in the region 180-200 of the KSHV-cyclin decreases the binding affinity to Cdk6 in U2OS cells but increases the activity of Cdk6.KSHV-cyclin complexes in vitro indicating that low affinity binding of cyclins to the Cdk subunit might favor increased on- or off-rates of Cdk substrates. Expression of high levels of p16(INK4a) in cells leads to the formation of a heterotrimeric complex composed of Cdk6, KSHV-cyclin, and p16(INK4a). Some of the Cdk6 .KSHV-cyclin.p16 complexes were found to be active indicating that there might be different modes of p16 binding to Cdk6.cyclin complexes.

Adenosine Triphosphate↗

PubMed2005

Activation of a nuclear Cdc2-related kinase within a mitogen-activated protein kinase-like TDY motif by autophosphorylation and cyclin-dependent protein kinase-activating kinase.

Male germ cell-associated kinase (MAK) and intestinal cell kinase (ICK) are nuclear Cdc2-related kinases with nearly identical N-terminal catalytic domains and more divergent C-terminal noncatalytic domains. The catalytic domain is also related to mitogen-activated protein kinases (MAPKs) and contains a corresponding TDY motif. Nuclear localization of ICK requires subdomain XI and interactions of the conserved Arg-272, but not kinase activity or, surprisingly, any of the noncatalytic domain. Further, nuclear localization of ICK is required for its activation. ICK is activated by dual phosphorylation of the TDY motif. Phosphorylation of Tyr-159 in the TDY motif requires ICK autokinase activity but confers only basal kinase activity. Full activation requires additional phosphorylation of Thr-157 in the TDY motif. Coexpression of ICK with constitutively active MEK1 or MEK5 fails to increase ICK phosphorylation or activity, suggesting that MEKs are not involved. ICK and MAK are related to Ime2p in budding yeast, and cyclin-dependent protein kinase-activating kinase Cak1p has been placed genetically upstream of Ime2p. Recombinant Cak1p phosphorylates Thr-157 in the TDY motif of recombinant ICK and activates its activity in vitro. Coexpression of ICK with wild-type CAK1 but not kinase-inactive CAK1 in cells also increases ICK phosphorylation and activity. Our studies establish ICK as the prototype for a new group of MAPK-like kinases requiring dual phosphorylation at TDY motifs.

Amino Acid Motifs↗

PubMed2005

Sil phosphorylation in a Pin1 binding domain affects the duration of the spindle checkpoint.

SIL is an immediate-early gene that is essential for embryonic development and is implicated in T-cell leukemia-associated translocations. We now show that the Sil protein is hyperphosphorylated during mitosis or in cells blocked at prometaphase by microtubule inhibitors. Cell cycle-dependent phosphorylation of Sil is required for its interaction with Pin1, a regulator of mitosis. Point mutation of the seven (S/T)P sites between amino acids 567 and 760 reduces mitotic phosphorylation of Sil, Pin1 binding, and spindle checkpoint duration. When a phosphorylation site mutant Sil is stably expressed, the duration of the spindle checkpoint is shortened in cells challenged with taxol or nocodazole, and the cells revert to a G2-like state. This event is associated with the downregulation of the kinase activity of the Cdc2/cyclin B1 complex and the dephosphorylation of the threonine 161 on the Cdc2 subunit. Sil downregulation by plasmid-mediated RNA interference limited the ability of cells to activate the spindle checkpoint and correlated with a reduction of Cdc2/cyclin B1 activity and phosphorylation on T161 on the Cdc2 subunit. These data suggest that a critical region of Sil is required to mediate the presentation of Cdc2 activity during spindle checkpoint arrest.

Binding Sites↗

PubMed2004

Lymphatic dysfunction in transgenic mice expressing KSHV k-cyclin under the control of the VEGFR-3 promoter.

Kaposi sarcoma-associated herpesvirus (KSHV) infects endothelial cells within KS tumors, and these cells express the KSHV latent-cycle gene k-cyclin (kCYC) as well as vascular endothelial growth factor receptor 3 (VEGFR-3), a marker for lymphatic endothelium. To further understand KSHV-mediated pathogenesis, we generated transgenic mice expressing kCYC under the control of the VEGFR-3 promoter. kCYC mRNA and functional protein expression within tissue correlated with VEGFR-3 expression and were most abundantly detected within lung tissue. Clinically, most transgenic mice died within 6 months of age secondary to progressive accumulation of chylous pleural fluid. In skin, edema was detected by magnetic resonance imaging and mice demonstrated persistent erythema of the ears following trauma. Histologically, erythematous skin showed extravasation of erythrocytes and accumulation of erythrocytes within lymphatic lumens. In addition, lymphatic drainage of injected contrast dyes was markedly impaired in transgenic mice. Karyomegaly, a feature observed in kCYC-expressing cells in vitro, was detected in many tissues, and selectively occurred within lymphatic endothelial cells expressing kCYC mRNA by in situ hybridization. In summary, kCYC expression within VEGFR-3+ cells of mice causes marked impairment of lymphatic function. kCYC may contribute to the development of certain clinical and histologic features of KS, including localized edema and retention of extravasated erythrocytes within KS tumors.

Animals↗