Regulation of Cdc25A half-life in interphase by cyclin-dependent kinase 2 activity

Cdc25A regulates cell cycle progression, has oncogenic and anti-apoptotic activity, and is over-expressed in many human tumors. Phosphorylation by Chk1 and Cds1/Chk2 down-regulates Cdc25A levels in response to genotoxic stresses. Nevertheless, it remains unclear whether Chk1 and Cds1/Chk2 are uniquely responsible for regulating Cdc25A stability during interphase or if other kinase activities contribute. Here we report that treatment of HeLa cells with the cyclin-dependent kinase inhibitor roscovitine caused a concentration- and time-dependent increase in Cdc25A protein levels. Transfection with dominant-negative Cdk mutants demonstrated that only a Cdk2 mutant increased Cdc25A protein levels; Cdk1 and Cdk3 mutants had no effect. The increased Cdc25A protein levels were the result of an increase in the half-life of the protein; no increase in Cdc25A mRNA levels was observed. These results demonstrate Cdk2 kinase activity contributes to the labile nature of Cdc25A during interphase and redefine the nature of the Cdc25A-Cdk2 autoamplification feedback loop.     

Regulation and role of cyclin-dependent kinase activity in neuronal survival and death

Cyclin-dependent kinase (Cdk)5 is a proline-directed Ser/Thr protein kinase that functions mainly in neurons and is activated by binding to a regulatory subunit, p35 or p39. Kinase activity is mainly determined by the amount of p35 available, which is controlled by a balance between synthesis and degradation. Kinase activity is also regulated by Cdk5 phosphorylation, but the activity of phosphorylated Cdk5 is in contrast to that of cycling Cdks. Cdk5 is a versatile protein kinase that regulates multiple neuronal activities including neuronal migration and synaptic signaling. Further, Cdk5 plays a role in both survival and death of neurons. Long-term inactivation of Cdk5 triggers cell death, and the survival activity of Cdk5 is apparent when neurons suffer from stress. In contrast, hyper-activation of Cdk5 by p25 promotes cell death, probably by reactivating cell-cycle machinery in the nucleus. The pro-death activity is suppressed by membrane association of Cdk5 via myristoylation of p35. Appropriate activity, localization, and regulation of Cdk5 may be critical for long-term survival of neurons, which is more than 80 years in the case of humans.     

Regulation of telomere elongation by the cyclin-dependent kinase CDK1

Telomere elongation is cell-cycle regulated and requires the coordinated activity of proteins involved in the DNA damage response. We used an assay that detects de novo telomere addition to examine the role of the cyclin-dependent kinase Cdk1 (Cdc28) in cell-cycle-specific telomere elongation. Inhibition of an ATP analog-sensitive allele of Cdk1 completely blocked the addition of telomere repeats. Mutations in Rif2 and DNA polymerase alpha that cause increased telomere elongation were unable to compensate for the loss of Cdk1 activity, suggesting Cdk1 activity is required for an early step in telomere addition. Mutations in DNA repair proteins that act with Cdk1 at double-strand breaks also prevented telomere elongation. Cdk1 activity was required for the generation of 3' single-strand overhangs at both native and de novo telomeres. We propose Cdk1 activity controls the timing of telomere elongation by regulating the single-strand overhang at chromosome ends.     

Regulation of protein phosphatase inhibitor-1 by cyclin-dependent kinase 5

Inhibitor-1, the first identified endogenous inhibitor of protein phosphatase 1 (PP-1), was previously reported to be a substrate for cyclin-dependent kinase 5 (Cdk5) at Ser67. Further investigation has revealed the presence of an additional Cdk5 site identified by mass spectrometry and confirmed by site-directed mutagenesis as Ser6. Basal levels of phospho-Ser6 inhibitor-1, as detected by a phosphorylation state-specific antibody against the site, existed in specific regions of the brain and varied with age. In the striatum, basal in vivo phosphorylation and dephosphorylation of Ser6 were mediated by Cdk5, PP-2A, and PP-1, respectively. Additionally, calcineurin contributed to dephosphorylation under conditions of high Ca2+. In biochemical assays the function of Cdk5-dependent phosphorylation of inhibitor-1 at Ser6 and Ser67 was demonstrated to be an intramolecular impairment of the ability of inhibitor-1 to be dephosphorylated at Thr35; this effect was recapitulated in two systems in vivo. Dephosphorylation of inhibitor-1 at Thr35 is equivalent to inactivation of the protein, as inhibitor-1 only serves as an inhibitor of PP-1 when phosphorylated by cAMP-dependent kinase (PKA) at Thr35. Thus, inhibitor-1 serves as a critical junction between kinase- and phosphatase-signaling pathways, linking PP-1 to not only PKA and calcineurin but also Cdk5.     

Regulation of ceramide biosynthesis by TOR complex 2

Ceramides and sphingoid long-chain bases (LCBs) are precursors to more complex sphingolipids and play distinct signaling roles crucial for cell growth and survival. Conserved reactions within the sphingolipid biosynthetic pathway are responsible for the formation of these intermediates. Components of target of rapamycin complex 2 (TORC2) have been implicated in the biosynthesis of sphingolipids in S. cerevisiae; however, the precise step regulated by this complex remains unknown. Here we demonstrate that yeast cells deficient in TORC2 activity are impaired for de novo ceramide biosynthesis both in vivo and in vitro. We find that TORC2 regulates this step in part by activating the AGC kinase Ypk2 and that this step is antagonized by the Ca2+/calmodulin-dependent phosphatase calcineurin. Because Ypk2 is activated independently by LCBs, the direct precursors to ceramides, our data suggest a model wherein TORC2 signaling is coupled with LCB levels to control Ypk2 activity and, ultimately, regulate ceramide formation.     

Regulation of the fusion pore conductance during exocytosis by cyclin-dependent kinase 5

Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase involved in synaptogenesis and brain development, and its enzymatic activity is essential for slow forms of synaptic vesicle endocytosis. Recent work also has implicated Cdk5 in exocytosis and synaptic plasticity. Pharmacological inhibition of Cdk5 modifies secretion in neuroendocrine cells, synaptosomes, and brain slices; however, the specific mechanisms involved remain unclear. Here we demonstrate that dominant-negative inhibition of Cdk5 increases quantal size and broadens the kinetics of individual exocytotic events measured by amperometry in adrenal chromaffin cells. Conversely, Cdk5 overexpression narrows the kinetics of fusion, consistent with an increase in the extent of kiss-and-run exocytosis. Cdk5 inhibition also increases the total charge and current of catecholamine released during the amperometric foot, representing a modification of the conductance of the initial fusion pore connecting the granule and plasma membrane. We suggest that these effects are not attributable to an alteration in catecholamine content of secretory granules and therefore represent an effect on the fusion mechanism itself. Finally, mutational silencing of the Cdk5 phosphorylation site in Munc18, an essential protein of the late stages of vesicle fusion, has identical effects on amperometric spikes as dominant-negative Cdk5 but does not affect the amperometric feet. Cells expressing Munc18 T574A have increased quantal size and broader kinetics of fusion. These results suggest that Cdk5 could, in part, control the kinetics of exocytosis through phosphorylation of Munc18, but Cdk5 also must have Munc18-independent effects that modify fusion pore conductance, which may underlie a role of Cdk5 in synaptic plasticity.     

Regulation of androgen receptor and prostate cancer growth by cyclin-dependent kinase 5

Prostate cancer is the most frequently diagnosed male malignancy. The normal prostate development and prostate cancer progression are mediated by androgen receptor (AR). Recently, the roles of cyclin-dependent kinase 5 (Cdk5) and its activator, p35, in cancer biology are explored one after another. We have previously demonstrated that Cdk5 may regulate proliferation of thyroid cancer cells. In addition, we also identify that Cdk5 overactivation can be triggered by drug treatments and leads to apoptosis of prostate cancer cells. The aim of this study is to investigate how Cdk5 regulates AR activation and growth of prostate cancer cells. At first, the data show that Cdk5 enables phosphorylation of AR at Ser-81 site through direct biochemical interaction and, therefore, results in the stabilization of AR proteins. The Cdk5-dependent AR stabilization causes accumulation of AR proteins and subsequent activation. Besides, the positive regulations of Cdk5-AR on cell growth are also determined in vitro and in vivo. S81A mutant of AR diminishes its interaction with Cdk5, reduces its nuclear localization, fails to stabilize its protein level, and therefore, decreases prostate cancer cell proliferation. Prostate carcinoma specimens collected from 177 AR-positive patients indicate the significant correlations between the protein levels of AR and Cdk5 or p35. These findings demonstrate that Cdk5 is an important modulator of AR and contributes to prostate cancer growth. Therefore, Cdk5-p35 may be suggested as diagnostic and therapeutic targets for prostate cancer in the near future.     

Regulation of ceramide channels by Bcl-2 family proteins

Mitochondrial outer membrane permeabilization to proteins, an irreversible step in apoptosis by which critical proteins are released, is tightly regulated by Bcl-2 family proteins. The exact nature of the release pathway is still undefined. Ceramide is an important sphingolipid, involved in various cellular processes including apoptosis. Here we describe the structural properties of ceramide channels and their regulation by the anti-apoptotic and pro-apoptotic proteins of the Bcl-2 family. The evolutionarily conserved regulation of ceramide channels by Bcl-2 family proteins, consistent with their role in apoptosis, lends credibility to the notion that ceramide channels constitute the protein release pathway.     

Regulation and traffic of ceramide 1-phosphate produced by ceramide kinase: comparative analysis to glucosylceramide and sphingomyelin

Ceramide 1-phosphate (C1P) has been characterized as a sphingolipid that participates in cell signaling. Although C1P synthesis is thought to occur via phosphorylation of ceramide by ceramide kinase (CerK), the processes that regulate C1P formation and fate remain largely unknown. In this study we analyzed bone marrow-derived macrophages (BMDM) from CerK-null mice (Cerk(-/-)) and found significant levels of C1P, suggesting that previously unrecognized pathways may also lead to C1P formation. After these experiments we used an overexpression system, BMDM from Cerk(-/-) mice, and short-chain fluorescent ceramides to trace CerK-dependent formation of C1P. Because the ceramide analogs can also be converted to glucosylceramide (GlcCer) and sphingomyelin (SM), they allowed us to directly compare all three metabolites. We found that C1P produced by CerK is turned over rapidly when serum is removed or upon calcium chelation, whereas GlcCer and SM are stable under these conditions. We further demonstrated that ceramide must be transported to the Golgi complex to be phosphorylated by CerK. Inhibition of the ceramide transfer protein slowed down SM formation without decreasing C1P, suggesting an alternate route of ceramide transport. Other experiments indicated that, like GlcCer and SM, C1P traffics along the secretory pathway to reach the plasma membrane. Furthermore, in BMDM C1P was secreted more readily than was GlcCer or SM. Altogether, our results indicate that CerK is essential to C1P formation via phosphorylation of Cer, providing the first insights into mechanisms underlying ceramide access to CerK and C1P trafficking as well as clarifying C1P as a signaling entity.     

Fibronectin receptor modulates cyclin-dependent kinase activity.

The high affinity fibronectin receptor (FNR) is expressed by hematopoietic cells, fibroblasts, and proliferating epidermal cells. Expression of this integrin is altered by chemical and viral transformation, suggesting that FNR dysfunction may play a role in growth control. This study demonstrates that exposing FA-K562 cells to glycine-arginine-glycine-aspartate-serine (GRGDS), a peptide ligand of the FNR, specifically stimulates p34/cdc2- and cyclin A-associated kinase activities. This occurs within 2 h of peptide addition. The 110-kDa form of the retinoblastoma protein appears within 3 h of GRGDS addition, consistent with activation of a G1/S kinase. DNA staining profiles demonstrate that GRGDS induces cell cycle progression within 24 h. Increased anchorage-independent growth is subsequently observed in GRGDS-treated FA-K562 cells. The control peptide, GRGES, which cannot bind the FNR, has none of these effects. This demonstrates that an extracellular integrin ligand can regulate cell proliferation. Furthermore, these results suggest that integrins link the extracellular environment and intracellular growth regulators.     

Regulation of yeast glycogen phosphorylase by the cyclin-dependent protein kinase Pho85p

Yeast accumulate glycogen in response to nutrient limitation. The key enzymes of glycogen synthesis and degradation, glycogen synthase, and phosphorylase, are regulated by reversible phosphorylation. Phosphorylation inactivates glycogen synthase but activates phosphorylase. The kinases and phosphatases that control glycogen synthase are well characterized whilst the enzymes modifying phosphorylase are poorly defined. Here, we show that the cyclin-dependent protein kinase, Pho85p, which we have previously found to regulate glycogen synthase also controls the phosphorylation state of phosphorylase.     

Regulation of phospholipase D2 activity by protein kinase C alpha

It has been well documented that protein kinase C (PKC) plays an important role in regulation of phospholipase D (PLD) activity. Although PKC regulation of PLD1 activity has been studied extensively, the role of PKC in PLD2 regulation remains to be established. In the present study it was demonstrated that phorbol 12-myristate 13-acetate (PMA) induced PLD2 activation in COS-7 cells. PLD2 was also phosphorylated on both serine and threonine residues after PMA treatment. PKC inhibitors Ro-31-8220 and bisindolylmaleimide I inhibited both PMA-induced PLD2 phosphorylation and activation. However, G? 6976, a PKC inhibitor relatively specific for conventional PKC isoforms, almost completely abolished PLD2 phosphorylation by PMA but only slightly inhibited PLD2 activation. Furthermore, time course studies showed that phosphorylation of PLD2 lagged behind its activation by PMA. Concentration curves for PMA action on PLD2 phosphorylation and activation also showed that PLD2 was activated by PMA at concentrations at which PMA didn't induce phosphorylation. A kinase-deficient mutant of PKCalpha stimulated PLD2 activity to an even higher level than wild type PKCalpha. Co-expression of wild type PKCalpha, but not PKCdelta, greatly enhanced both basal and PMA-induced PLD2 phosphorylation. A PKCdelta-specific inhibitor, rottlerin, failed to inhibit PMA-induced PLD2 phosphorylation and activation. Co-immunoprecipitation studies indicated an association between PLD2 and PKCalpha under basal conditions that was further enhanced by PMA. Time course studies of the effects of PKCalpha on PLD2 showed that as the phosphorylation of PLD2 increased, its activity declined. In summary, the data demonstrated that PLD2 is activated and phosphorylated by PMA and PKCalpha in COS-7 cells. However, the phosphorylation is not required for PKCalpha to activate PLD2. It is suggested that interaction rather than phosphorylation underscores the activation of PLD2 by PKC in vivo and that phosphorylation may contribute to the inactivation of the enzyme.     

Modulation of beta-catenin phosphorylation/degradation by cyclin-dependent kinase 2

beta-Catenin functions as a downstream component of the Wnt/Wingless signal transduction pathway, and inappropriate control of cytosolic beta-catenin is a crucial step in the genesis of several human cancers. Here we demonstrate that cyclin-dependent kinase 2 (CDK2) in association with cyclin A or cyclin E directly binds to beta-catenin. In vivo and in vitro kinase assays with cyclin-CDK2 demonstrate beta-catenin phosphorylation on residues Ser(33), Ser(37), Thr(41), and Ser(45). This phosphorylation promotes rapid degradation of cytosolic beta-catenin via the beta-TrCP-mediated proteasome pathway. Moreover, cyclin E-CDK2 contributes to rapid degradation of cytosolic beta-catenin levels during G(1) phase by regulating beta-catenin phosphorylation and subsequent degradation. In this way, CDK2 may "fine tune" beta-catenin levels over the course of the cell cycle.     

Regulation of secretory transport by protein kinase D-mediated phosphorylation of the ceramide transfer protein

Protein kinase D (PKD) has been identified as a crucial regulator of secretory transport at the trans-Golgi network (TGN). Recruitment and activation of PKD at the TGN is mediated by the lipid diacylglycerol, a pool of which is generated by sphingomyelin synthase from ceramide and phosphatidylcholine. The nonvesicular transfer of ceramide from the endoplasmic reticulum to the Golgi complex is mediated by the lipid transfer protein CERT (ceramide transport). In this study, we identify CERT as a novel in vivo PKD substrate. Phosphorylation on serine 132 by PKD decreases the affinity of CERT toward its lipid target phosphatidylinositol 4-phosphate at Golgi membranes and reduces ceramide transfer activity, identifying PKD as a regulator of lipid homeostasis. We also show that CERT, in turn, is critical for PKD activation and PKD-dependent protein cargo transport to the plasma membrane. Thus, the interdependence of PKD and CERT is key to the maintenance of Golgi membrane integrity and secretory transport.