Alternative splicing in the regulatory region of the human phosphatases CDC25A and CDC25C

CDC25 phosphatases play key roles in cell proliferation by activating cell cycle-specific cyclin-dependent kinases (CDKs). We identified four new splice variants in the amino-terminal regulatory region of human cdc25C and one in cdc25A. All variants except one retain an intact catalytic domain. Alternative splicing results in loss of phosphorylation sites for kinases like CDK and the calcium/calmodulin-dependent kinase II (CaMKII), which influence CDC25 activity and compartmental localization. In NT2 teratocarcinoma cells, induced for nerve cell differentiation, the smaller sized variant of cdc25C was upregulated. At the protein level both phosphorylation state and isoform distribution differed between cell lines and cell cycle phases.     

A novel GRK2/HDAC6 interaction modulates cell spreading and motility

Cell motility and adhesion involves dynamic microtubule (MT) acetylation/deacetylation, a process regulated by enzymes as HDAC6, a major cytoplasmic α-tubulin deacetylase. We identify G protein-coupled receptor kinase 2 (GRK2) as a key novel stimulator of HDAC6. GRK2, which levels inversely correlate with the extent of α-tubulin acetylation in epithelial cells and fibroblasts, directly associates with and phosphorylates HDAC6 to stimulate α-tubulin deacetylase activity. Remarkably, phosphorylation of GRK2 itself at S670 specifically potentiates its ability to regulate HDAC6. GRK2 and HDAC6 colocalize in the lamellipodia of migrating cells, leading to local tubulin deacetylation and enhanced motility. Consistently, cells expressing GRK2-K220R or GRK2-S670A mutants, unable to phosphorylate HDAC6, exhibit highly acetylated cortical MTs and display impaired migration and protrusive activity. Finally, we find that a balanced, GRK2/HDAC6-mediated regulation of tubulin acetylation differentially modulates the early and late stages of cellular spreading. This novel GRK2/HDAC6 functional interaction may have important implications in pathological contexts.     

NPAS2 regulates proliferation of acute myeloid leukemia cells via CDC25A-mediated cell cycle progression and apoptosis

Promoted proliferation and associated suppression of apoptosis at various stages of myeloid differentiation are well-known features of acute myeloid leukemia (AML), but understanding of the molecular processes involved remains limited. As a crucial circadian agent, neuronal PAS domain protein 2 (NPAS2) is widely recognized as a promising predictor of clinical outcome in various malignancies. Nevertheless, the understanding of its influence on AML is insufficient. Using KD cells and expression assays, we carried out detailed investigation of the role of NPAS2 in AML in vivo and in vitro. Firstly, we found that NPAS2 expression was elevated in AML cells both in vivo and in vitro. NPAS2 knockdown via lentiviral infection clearly suppressed proliferation of MV4-11 and MOLM-14 cells. Additionally, NPAS2 knockdown caused G1/S cell cycle arrest (CCA), which inhibited CDC25A expression. Moreover, NPAS2 knockdown promoted cell death, as evidenced by increased caspase-3 cleavage, and change in Bcl2/Bax production. Excessive CDC25A expression eliminated G1/S CCA triggered by NPAS2 knockdown and death of NPAS2 knocked down MOLM and MV4-11 cells. The expression of CDC25A was stabilized by NPAS2, which induced cell cycle progression and participated in suppression of cell death by modulating caspase-3 cleavage, and expression of Bcl2/Bax. We therefore indicated NPAS2 to be a crucial modulator of survival as well as proliferation. Our research sheds light on the etiology of the proliferation of promyelocytes modulated via NPAS2 with regard to AML.     

A lipocalin sequence signature modulates cell survival

Lipocalins are β-barrel proteins, which share three conserved motifs in their amino acid sequence. In this study, we identified by a peptide mapping approach, a seven-amino acid sequence related to one of these motifs (motif 2) that modulates cell survival. A synthetic peptide based on an insect lipocalin displayed cytoprotective activity in serum-deprived endothelial cells and leucocytes. This activity was dependent on nitric oxide synthase. This sequence was found within several lipocalins, including apolipoprotein D, retinol binding protein, lipocalin-type prostaglandin D synthase, and many unknown proteins, suggesting that it is a sequence signature and a lipocalin conserved property.     

Lin-28 homologue A (LIN28A) promotes cell cycle progression via regulation of cyclin-dependent kinase 2 (CDK2), cyclin D1 (CCND1), and cell division cycle 25 homolog A (CDC25A) expression in cancer

The RNA-binding protein LIN28A regulates the translation and stability of a large number of mRNAs as well as the biogenesis of certain miRNAs in embryonic stem cells and developing tissues. Increasing evidence indicates that LIN28A functions as an oncogene promoting cancer cell growth. However, little is known about its molecular mechanism of cell cycle regulation in cancer. Using tissue microarrays, we found that strong LIN28A expression was reactivated in about 10% (7.1-17.1%) of epithelial tumors (six tumor types, n = 369). Both in vitro and in vivo experiments demonstrate that LIN28A promotes cell cycle progression in cancer cells. Genome-wide RNA-IP-chip experiments indicate that LIN28A binds to thousands of mRNAs, including a large group of cell cycle regulatory mRNAs in cancer and embryonic stem cells. Furthermore, the ability of LIN28A to stimulate translation of LIN28A-binding mRNAs, such as CDK2, was validated in vitro and in vivo. Finally, using a combined gene expression microarray and bioinformatics approach, we found that LIN28A also regulates CCND1 and CDC25A expression and that this is mediated by inhibiting the biogenesis of let-7 miRNA. Taken together, these results demonstrate that LIN28A is reactivated in about 10% of epithelial tumors and promotes cell cycle progression by regulation of both mRNA translation (let-7-independent) and miRNA biogenesis (let-7-dependent).     

Cyclin D1 serves as a cell cycle regulatory switch in actively proliferating cells

Much of our current understanding of the cell cycle involves analyses of its induction in quiescent cells. To better understand the control of cell cycle propagation and termination, studies have been performed in actively cycling cultures using time-lapse photography and quantitative image analysis. These studies reveal a highly ordered sequence of events required for promotion of continued proliferation. The decision to continue cell cycle progression takes place in G2 phase, when cellular Ras induces the elevation of cyclin D1 levels. These levels are maintained through G1 phase and are required for the initiation of S phase, at which time cyclin D1 levels are automatically reduced to low levels. The reduction of cyclin D1 to low levels during S phase is required for DNA synthesis, and forces the cell to induce high cyclin D1 levels once again when it enters G2 phase. In this way, cyclin D1 is proposed to serve as an active switch in the regulation of continued cell cycle progression.     

1alpha,25(OH)2-vitamin D3 membrane-initiated calcium signaling modulates exocytosis and cell survival

1alpha,25(OH)(2)-vitamin D(3) (1,25D) is considered a bone anabolic hormone. 1,25D actions leading to bone formation involve gene transactivation, on one hand, and modulation of cytoplasmic signaling, on the other. In both cases, a functional vitamin D receptor (VDR) appears to be required. Here we study 1,25D-stimulated calcium signaling that initiates at the cell membrane and leads to exocytosis of bone materials and increased osteoblast survival. We found that rapid 1,25D-induction of exocytosis couples to cytoplasmic calcium increase in osteoblastic ROS 17/2.8 cells. In addition, we found that elevation of cytoplasmic calcium concentration is involved in 1,25D anti-apoptotic effects via Akt activation in ROS 17/2.8 cells and non-osteoblastic CV-1 cells. In both cases, 1,25D-stimulated elevation of intracellular calcium is due in part to activation of L-type Ca(2+) channels. We conclude that 1,25D bone anabolic effects that involve increased intracellular Ca(2+) concentration in osteoblasts can be explained at two levels. At the single-cell level, 1,25D promotes Ca(2+)-dependent exocytotic activities. At the tissue level, 1,25D protects osteoblasts from apoptosis via a Ca(2+)-dependent Akt pathway. Our studies contribute to the understanding of the molecular basis of bone diseases characterized by decreased bone formation and mineralization.     

Cdc25A is a novel phosphatase functioning early in the cell cycle.

The cdc25+ tyrosine phosphatase is a key mitotic inducer of the fission yeast Schizosaccharomyces pombe, controlling the timing of the initiation of mitosis. Mammals contain at least three cdc25+ homologues called cdc25A, cdc25B and cdc25C. In this study we investigate the biological function of cdc25A. Although very potent in rescuing the S.pombe cdc25 mutant, cdc25A is less structurally related to the S.pombe enzyme. Northern and Western blotting detection reveals that unlike cdc25B, cdc25C and cdc2, cdc25A is predominantly expressed in late G1. Moreover, immunodepletion of cdc25A in rat cells by microinjection of a specific antibody effectively blocks their cell cycle progression from G1 into the S phase, as determined by laser scanning single cell cytometry. These results indicate that cdc25A is not a mitotic regulator but a novel phosphatase that plays a crucial role in the start of the cell cycle. In view of its strong ability to activate cdc2 kinase and its specific expression in late G1, cdc2-related kinases functioning early in the cell cycle may be targets for this phosphatase.     

Sulforaphane-induced G2/M phase cell cycle arrest involves checkpoint kinase 2-mediated phosphorylation of cell division cycle 25C

Previously, we showed that sulforaphane (SFN), a naturally occurring cancer chemopreventive agent, effectively inhibits proliferation of PC-3 human prostate cancer cells by causing caspase-9- and caspase-8-mediated apoptosis. Here, we demonstrate that SFN treatment causes an irreversible arrest in the G(2)/M phase of the cell cycle. Cell cycle arrest induced by SFN was associated with a significant decrease in protein levels of cyclin B1, cell division cycle (Cdc) 25B, and Cdc25C, leading to accumulation of Tyr-15-phosphorylated (inactive) cyclin-dependent kinase 1. The SFN-induced decline in Cdc25C protein level was blocked in the presence of proteasome inhibitor lactacystin, but lactacystin did not confer protection against cell cycle arrest. Interestingly, SFN treatment also resulted in a rapid and sustained phosphorylation of Cdc25C at Ser-216, leading to its translocation from the nucleus to the cytoplasm because of increased binding with 14-3-3beta. Increased Ser-216 phosphorylation of Cdc25C upon treatment with SFN was the result of activation of checkpoint kinase 2 (Chk2), which was associated with Ser-1981 phosphorylation of ataxia telangiectasia-mutated, generation of reactive oxygen species, and Ser-139 phosphorylation of histone H2A.X, a sensitive marker for the presence of DNA double-strand breaks. Transient transfection of PC-3 cells with Chk2-specific small interfering RNA duplexes significantly attenuated SFN-induced G(2)/M arrest. HCT116 human colon cancer-derived Chk2(-/-) cells were significantly more resistant to G(2)/M arrest by SFN compared with the wild type HCT116 cells. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in irreversible G(2)/M arrest by SFN. Activation of Chk2 in response to DNA damage is well documented, but the present study is the first published report to link Chk2 activation to cell cycle arrest by an isothiocyanate.     

A tale of two components: a novel kinase and a regulatory switch

Histidine protein kinases and response regulators form the basis of phosphotransfer signal transduction pathways. Commonly referred to as two-component systems, these modular and adaptable signaling schemes are prevalent in prokaryotes. Structures of the core domains of histidine kinases reveal a protein kinase fold different from that of the Ser/Thr/Tyr protein kinase family, but similar to that of other ATP binding domains. Recent structure determinations of phosphorylated response regulator domains indicate a conserved mechanism for the propagated conformational change that accompanies phosphorylation of an active site Asp residue. The altered molecular surface promotes specific protein-protein interactions that mediate the downstream response.     

A murine CDC25/ras-GRF-related protein implicated in ras regulation

A partial cDNA encoding a novel putative p2, ras guanine nucleotide release-inducing factor (GRF), GRF2, was amplified from murine embryonic stem cells. The presumptive catalytic region of GRF2 is related to the yeast Ras GRF encoded by CDC25. GRF2 is 80% identical to murine CDC25Mm/ras-GRF, but is more similar to yeast CDC25 than to other ras GRFs related to the Drosophila son of sevenless gene product. A 9-kb GRF2 messenger RNA was highly expressed in brain, but GRF2-specific antibodies recognized apparent GRF2 proteins in various mouse tissues in addition to brain. Thus GRF2 represents a novel widely-expressed protein that is highly related to CDC25Mm/ras-GRF, at least in its catalytic domain. Both GRF2 and CDC25Mm/ras-GRF are expressed in murine embryonic stem cells, suggesting that different Ras activators may regulate ras-dependent proliferation and differentiation in early mouse development. © 1993Wiley-Liss, Inc.     

DYRK3 activation,engagement of protein kinase A/cAMP response element-binding protein,and modulation of progenitor cell survival

DYRKs are a new family of dual-specificity tyrosine-regulated kinases with emerging roles in cell growth and development. Recently, we discovered that DYRK3 is expressed primarily in erythroid progenitor cells and modulates late erythropoiesis. We now describe 1) roles for the DYRK3 YTY signature motif in kinase activation, 2) the coupling of DYRK3 to cAMP response element (CRE)-binding protein (CREB), and 3) effects of DYRK3 on hematopoietic progenitor cell survival. Regarding the DYRK3 kinase domain, intactness of Tyr(333) (but not Tyr(331)) within subdomain loop VII-VIII was critical for activation. Tyr(331) plus Tyr(333) acidification (Tyr mutated to Glu) was constitutively activating, but kinase activity was not affected substantially by unique N- or C-terminal domains. In transfected 293 and HeLa cells, DYRK3 was discovered to efficiently stimulate CRE-luciferase expression, to activate a CREB-Gal4 fusion protein, and to promote CREB phosphorylation at Ser(133). Interestingly, this CREB/CRE response was also supported (50% of wild-type activity) by a kinase-inactive DYRK3 mutant as well as a DYRK3 C-terminal region and was blocked by protein kinase A inhibitors, suggesting functional interactions between protein kinase A and DYRK3. Finally, DYRK3 expression in cytokine-dependent hematopoietic FDCW2 cells was observed to inhibit programmed cell death. Thus, primary new insight into DYRK3 kinase signaling routes, subdomain activities, and possible biofunctions is provided.     

Human Cdc14A phosphatase modulates the G2/M transition through Cdc25A and Cdc25B

The Cdc14 family of serine-threonine phosphatases antagonizes CDK activity by reversing CDK-dependent phosphorylation events. It is well established that the yeast members of this family bring about the M/G1 transition. Budding yeast Cdc14 is essential for CDK inactivation at the end of mitosis and fission yeast Cdc14 homologue Flp1/Clp1 down-regulates Cdc25 to ensure the inactivation of mitotic CDK complexes to trigger cell division. However, the functions of human Cdc14 homologues remain poorly understood. Here we have tested the hypothesis that Cdc14A might regulate Cdc25 mitotic inducers in human cells. We found that increasing levels of Cdc14A delay entry into mitosis by inhibiting Cdk1-cyclin B1 activity. By contrast, lowering the levels of Cdc14A accelerates mitotic entry. Biochemical analyses revealed that Cdc14A acts through key Cdk1-cyclin B1 regulators. We observed that Cdc14A directly bound to and dephosphorylated Cdc25B, inhibiting its catalytic activity. Cdc14A also regulated the activity of Cdc25A at the G2/M transition. Our results indicate that Cdc14A phosphatase prevents premature activation of Cdk1 regulating Cdc25A and Cdc25B at the entry into mitosis.     

Phosphorylation of Bcl-2 protein by CDC2 kinase during G2/M phases and its role in cell cycle regulation

Although it has been reported that Bcl-2 phosphorylation is associated with certain types of apoptosis, there is much controversy over the functional significance of and the kinases responsible for the phosphorylation. In this study, we examined whether Bcl-2 is phosphorylated by CDC2 kinase, a master regulator of G(2)/M transition in the eukaryotic cell cycle. When CDC2 was activated by okadaic acid in HL-60 cells, Bcl-2 phosphorylation was readily induced. The phosphorylation was correlated with the accumulation of cells in G(2)/M phases, but was not proportional to the level of apoptosis. Furthermore, we found that Bcl-2 was phosphorylated during G(2)/M phases of normal cell cycle. The ability of CDC2 to phosphorylate Bcl-2 was confirmed by in vitro kinase assay with a highly purified CDC2-cyclin B complex. Using synthetic peptides and mutant cell lines, we identified threonine 56, one of two consensus sites for CDC2 within the Bcl-2 sequence, as a residue phosphorylated by CDC2. Mutation at threonine 56 abrogated the cell cycle inhibitory effect of Bcl-2 without affecting anti-apoptotic function. These results suggest that two distinct functions of Bcl-2 (anti-apoptosis and cell cycle inhibition) are differentially regulated by post-translational mechanisms such as phosphorylation. CDC2-mediated phosphorylation of Bcl-2 may play some physiological roles in the negative regulatory events during mitosis.