Cyclin T1 expression is regulated by multiple signaling pathways and mechanisms during activation of human peripheral blood lymphocytes

Stimulation of primary human T lymphocytes results in up-regulation of cyclin T1 expression, which correlates with phosphorylation of the C-terminal domain of RNA polymerase II (RNAP II). Up-regulation of cyclin T1 and concomitant stabilization of cyclin-dependent kinase 9 (CDK9) may facilitate productive replication of HIV in activated T cells. We report that treatment of PBLs with two mitogens, PHA and PMA, results in accumulation of cyclin T1 via distinct mechanisms. PHA induces accumulation of cyclin T1 mRNA and protein, which results from cyclin T1 mRNA stabilization, without significant change in cyclin T1 promoter activity. Cyclin T1 mRNA stabilization requires the activation of both calcineurin and JNK because inhibition of either precludes cyclin T1 accumulation. In contrast, PMA induces cyclin T1 protein up-regulation by stabilizing cyclin T1 protein, apparently independently of the proteasome and without accumulation of cyclin T1 mRNA. This process is dependent on Ca2+-independent protein kinase C activity but does not require ERK1/2 activation. We also found that PHA and anti-CD3 Abs induce the expression of both the cyclin/CDK complexes involved in RNAP II C-terminal domain phosphorylation and the G1-S cyclins controlling cell cycle progression. In contrast, PMA alone is a poor inducer of the expression of G1-S cyclins but often as potent as PHA in inducing RNAP II cyclin/CDK complexes. These findings suggest coordination in the expression and activation of RNAP II kinases by pathways that independently stimulate gene expression but are insufficient to induce S phase entry in primary T cells.     

Identification and characterization of the CDK12/cyclin L1 complex involved in alternative splicing regulation

CrkRS is a Cdc2-related protein kinase that contains an arginine- and serine-rich (SR) domain, a characteristic of the SR protein family of splicing factors, and is proposed to be involved in RNA processing. However, whether it acts together with a cyclin and at which steps it may function to regulate RNA processing are not clear. Here, we report that CrkRS interacts with cyclin L1 and cyclin L2, and thus rename it as the long form of cyclin-dependent kinase 12 (CDK12(L)). A shorter isoform of CDK12, CDK12(S), that differs from CDK12(L) only at the carboxyl end, was also identified. Both isoforms associate with cyclin L1 through interactions mediated by the kinase domain and the cyclin domain, suggesting a bona fide CDK/cyclin partnership. Furthermore, CDK12 isoforms alter the splicing pattern of an E1a minigene, and the effect is potentiated by the cyclin domain of cyclin L1. When expression of CDK12 isoforms is perturbed by small interfering RNAs, a reversal of the splicing choices is observed. The activity of CDK12 on splicing is counteracted by SF2/ASF and SC35, but not by SRp40, SRp55, and SRp75. Together, our findings indicate that CDK12 and cyclin L1/L2 are cyclin-dependent kinase and cyclin partners and regulate alternative splicing.     

Activation of human cyclin-dependent kinases in vitro.

We have analyzed the activation of human cyclin-dependent kinases in a cell-free system. Human CDC2, cyclin-dependent kinase 2 (CDK2), cyclin A, and cyclin B1 were produced in insect cells by infection with recombinant baculoviruses. CDC2 or CDK2 monomers in lysates of infected cells could be activated by the addition of lysates containing cyclin A or B1. CDC2 activation by cyclin B1, as well as CDK2 activation by cyclins A and B1, was accompanied by the formation of high molecular weight complexes. In contrast, CDC2 did not bind effectively to cyclin A. CDC2 activation by cyclin B1 was studied in detail and was found to be accompanied by phosphorylation of CDC2 on Threonine 161. The binding of CDC2 to cyclin B1 also occurred under conditions where CDC2 phosphorylation was prevented, resulting in an inactive complex that could then be phosphorylated and activated on addition of cell extract. Highly purified CDC2 and cyclin B1 also formed inactive complexes that could be activated in an ATP-dependent fashion by unidentified components in crude cell extracts. These data suggest that the CDC2 activation process begins with cyclin binding, after which CDC2 phosphorylation, catalyzed by a separate enzyme, leads to activation.     

Activation of Transient Receptor Potential Vanilloid 4 Promotes the Proliferation of Stem Cells in the Adult Hippocampal Dentate Gyrus

Neurogenesis plays an important role in adult hippocampal function, and this process can be modulated by intracellular calcium. The activation of transient receptor potential vanilloid 4 (TRPV4) induces an increase in intracellular calcium concentration, but whether neurogenesis can be modulated by TRPV4 activation remains unclear. Here, we report that intracerebroventricular injection of the TRPV4 agonist GSK1016790A for 5 days enhanced the proliferation of stem cells in the hippocampal dentate gyrus (DG) of adult mice without affecting neurite growth, differentiation, or survival of newborn cells. GSK1016790A induced increases in the hippocampal protein levels of cyclin-dependent kinase (CDK) 6, CDK2, cyclin E1, and cyclin A2 but did not affect CDK4 and cyclin D1 expression. The phosphorylation of retinoblastoma protein (Rb) in hippocampi was enhanced in GSK1016790A-injected mice compared with control mice. Moreover, hippocampal protein levels of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation were enhanced by GSK1016790A. Finally, GSK1016790A-enhanced proliferation was markedly blocked by a MAPK/ERK kinase or p38 MAPK antagonist (U0126 or SB203580, respectively). The increased protein levels of CDK2 and CDK6, as well as those of cyclin E1 and cyclin A2, in GSK1016790A-injected mice were substantially reduced by co-injection of U0126 or SB203580. We conclude that TRPV4 activation results in the proliferation of stem cells in the adult hippocampal DG, which is likely mediated through ERK1/2 and p38 MAPK signaling to increase the expression of CDKs (CDK6 and CDK2) and cyclins (cyclin E1 and A2), phosphorylate Rb consequently, and accelerate the cell cycle ultimately.     

Xenopus phospho-CDK7/cyclin H expressed in baculoviral-infected insect cells.

The cyclin-dependent kinase-activating kinase (CAK) catalyzes the phosphorylation of the cyclin-dependent protein kinases (CDKs) on a threonine residue (Thr160 in human CDK2). The reaction is an obligatory step in the activation of the CDKs. In higher eukaryotes, the CAK complex has been characterized in two forms. The first consists of three subunits, namely CDK7, cyclin H, and an assembly factor called MAT1, while the second consists of phospho-CDK7 and cyclin H. Phosphorylation of CDK7 is essential for cyclin association and kinase activity in the absence of the assembly factor MAT1. The Xenopus laevis CDK7 phosphorylation sites are located on the activation segment of the kinase at residues Ser170 and at Thr176 (the latter residue corresponding to Thr160 in human CDK2). We report the expression and purification of X. laevis CDK7/cyclin H binary complex in insect cells through coinfection with the recombinant viruses, AcCDK7 and Accyclin H. Quantities suitable for crystallization trials have been obtained. The purified CDK7/cyclin H binary complex phosphorylated CDK2 and CDK2/cyclin A but did not phosphorylate histone H1 or peptide substrates based on the activation segments of CDK7 and CDK2. Analysis by mass spectrometry showed that coexpression of CDK7 with cyclin H in baculoviral-infected insect cells results in phosphorylation of residues Ser170 and Thr176 in CDK7. It is assumed that phosphorylation is promoted by kinase(s) in the insect cells that results in the correct, physiologically significant posttranslational modification. We discuss the occurrence of in vivo phosphorylation of proteins expressed in baculoviral-infected insect cells.     

Regulation of Exit from Quiescence by p27 and Cyclin D1-CDK4

The synthesis of cyclin D1 and its assembly with cyclin-dependent kinase 4 (CDK4) to form an active complex is a rate-limiting step in progression through the G₁ phase of the cell cycle. Using an activated allele of mitogen-activated protein kinase kinase 1 (MEK1), we show that this kinase plays a significant role in positively regulating the expression of cyclin D1. This was found both in quiescent serum-starved cells and in cells expressing dominant-negative Ras. Despite the observation that cyclin D1 is a target of MEK1, in cycling cells, activated MEK1, but not cyclin D1, is capable of overcoming a G₁ arrest induced by Ras inactivation. Either wild-type or catalytically inactive CDK4 cooperates with cyclin D1 in reversing the G₁ arrest induced by inhibition of Ras activity. In quiescent NIH 3T3 cells expressing either ectopic cyclin D1 or activated MEK1, cyclin D1 is able to efficiently associate with CDK4; however, the complex is inactive. A significant percentage of the cyclin D1-CDK4 complexes are associated with p27 in serum-starved activated MEK1 or cyclin D1 cell lines. Reduction of p27 levels by expression of antisense p27 allows for S-phase entry from quiescence in NIH 3T3 cells expressing ectopic cyclin D1, but not in parental cells.     

Structural basis for the autoinhibition and STI-571 inhibition of c-Kit tyrosine kinase

The activity of the c-Kit receptor protein-tyrosine kinase is tightly regulated in normal cells, whereas deregulated c-Kit kinase activity is implicated in the pathogenesis of human cancers. The c-Kit juxtamembrane region is known to have an autoinhibitory function; however the precise mechanism by which c-Kit is maintained in an autoinhibited state is not known. We report the 1.9-A resolution crystal structure of native c-Kit kinase in an autoinhibited conformation and compare it with active c-Kit kinase. Autoinhibited c-Kit is stabilized by the juxtamembrane domain, which inserts into the kinase-active site and disrupts formation of the activated structure. A 1.6-A crystal structure of c-Kit in complex with STI-571 (Imatinib or Gleevec) demonstrates that inhibitor binding disrupts this natural mechanism for maintaining c-Kit in an autoinhibited state. Together, these results provide a structural basis for understanding c-Kit kinase autoinhibition and will facilitate the structure-guided design of specific inhibitors that target the activated and autoinhibited conformations of c-Kit kinase.     

Activation mechanism of solubilized epidermal growth factor receptor tyrosine kinase.

Dimerization of epidermal growth factor receptor (EGFR) leads to the activation of its tyrosine kinase. To elucidate whether dimerization is responsible for activation of the intracellular tyrosine kinase domain or just plays a role in the stabilization of the active form, the activated status of wild-type EGFR moiety in the heterodimer with kinase activity-deficient mutant receptors was investigated. The kinase activity of the wild-type EGFR was partially activated by EGF in the heterodimer with intracellular domain deletion (sEGFR) or ATP binding-deficient mutant (K721A) EGFRs, while the wild-type EGFR in the heterodimer of wild-type and phosphate transfer activity-deficient mutant receptor D813N could be fully activated. After treatment with EGF, the ATP binding affinity and the V(max) of the wild-type EGFR increased. In the presence of sEGFR, a similar increase in the affinity for ATP was observed, but V(max) did not change. A two-step activation mechanism for EGFR was proposed: upon binding of EGF, the affinity for ATP increased and then, as a result of interaction between the neighboring tyrosine kinase domain, V(max) increased.     

Phosphoprotein-protein interactions revealed by the crystal structure of kinase-associated phosphatase in complex with phosphoCDK2.

The CDK-interacting protein phosphatase KAP dephosphorylates phosphoThr-160 (pThr-160) of the CDK2 activation segment, the site of regulatory phosphorylation that is essential for kinase activity. Here we describe the crystal structure of KAP in association with pThr-160-CDK2, representing an example of a protein phosphatase in complex with its intact protein substrate. The major protein interface between the two molecules is formed by the C-terminal lobe of CDK2 and the C-terminal helix of KAP, regions remote from the kinase-activation segment and the KAP catalytic site. The kinase-activation segment interacts with the catalytic site of KAP almost entirely via the phosphate group of pThr-160. This interaction requires that the activation segment is unfolded and drawn away from the kinase molecule, inducing a conformation of CDK2 similar to the activated state observed in the CDK2/cyclin A complex.     

PCTAIRE Kinase 3/Cyclin-dependent Kinase 18 Is Activated through Association with Cyclin A and/or Phosphorylation by Protein Kinase A

PCTAIRE kinase 3 (PCTK3)/cyclin-dependent kinase 18 (CDK18) is an uncharacterized member of the CDK family because its activator(s) remains unidentified. Here we describe the mechanisms of catalytic activation of PCTK3 by cyclin A2 and cAMP-dependent protein kinase (PKA). Using a pulldown experiment with HEK293T cells, cyclin A2 and cyclin E1 were identified as proteins that interacted with PCTK3. An in vitro kinase assay using retinoblastoma protein as the substrate showed that PCTK3 was specifically activated by cyclin A2 but not by cyclin E1, although its activity was lower than that of CDK2. Furthermore, immunocytochemistry analysis showed that PCTK3 colocalized with cyclin A2 in the cytoplasm and regulated cyclin A2 stability. Amino acid sequence analysis revealed that PCTK3 contained four putative PKA phosphorylation sites. In vitro and in vivo kinase assays showed that PCTK3 was phosphorylated by PKA at Ser¹², Ser⁶⁶, and Ser¹⁰⁹ and that PCTK3 activity significantly increased via phosphorylation at Ser¹² by PKA even in the absence of cyclin A2. In the presence of cyclin A2, PCTK3 activity was comparable to CDK2 activity. We also found that PCTK3 knockdown in HEK293T cells induced polymerized actin accumulation in peripheral areas and cofilin phosphorylation. Taken together, our results provide the first evidence for the mechanisms of catalytic activation of PCTK3 by cyclin A2 and PKA and a physiological function of PCTK3.     

Ringo/cyclin-dependent kinase and mitogen-activated protein kinase signaling pathways regulate the activity of the cell fate determinant Musashi to promote cell cycle re-entry in Xenopus oocytes

Cell cycle re-entry during vertebrate oocyte maturation is mediated through translational activation of select target mRNAs, culminating in the activation of mitogen-activated protein kinase and cyclin B/cyclin-dependent kinase (CDK) signaling. The temporal order of targeted mRNA translation is crucial for cell cycle progression and is determined by the timing of activation of distinct mRNA-binding proteins. We have previously shown in oocytes from Xenopus laevis that the mRNA-binding protein Musashi targets translational activation of early class mRNAs including the mRNA encoding the Mos proto-oncogene. However, the molecular mechanism by which Musashi function is activated is unknown. We report here that activation of Musashi1 is mediated by Ringo/CDK signaling, revealing a novel role for early Ringo/CDK function. Interestingly, Musashi1 activation is subsequently sustained through mitogen-activated protein kinase signaling, the downstream effector of Mos mRNA translation, thus establishing a positive feedback loop to amplify Musashi function. The identified regulatory sites are present in mammalian Musashi proteins, and our data suggest that phosphorylation may represent an evolutionarily conserved mechanism to control Musashi-dependent target mRNA translation.     

Reciprocal activation by cyclin-dependent kinases 2 and 7 is directed by substrate specificity determinants outside the T loop

Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the metazoan CDK-activating kinase (CAK), which activates CDKs, such as CDC2 and CDK2, through phosphorylation of a conserved threonine residue in the T loop. Full activation of CDK7 requires association with a positive regulatory subunit, cyclin H, and phosphorylation of a conserved threonine residue at position 170 in its own T loop. We show that threonine-170 of CDK7 is phosphorylated in vitro by its targets, CDC2 and CDK2, which also phosphorylate serine-164 in the CDK7 T loop, a site that perfectly matches their consensus phosphorylation site. In contrast, neither CDK4 nor CDK7 itself can phosphorylate the CDK7 T loop in vitro. The ability of CDC2 or CDK2 and CDK7 to phosphorylate each other but not themselves implies that each kinase can discriminate among closely related sequences and can recognize a substrate site that diverges from its usual preferred site. To understand the basis for this paradoxical substrate specificity, we constructed a chimeric CDK with the T loop of CDK7 grafted onto the body of CDK2. Surprisingly, the hybrid enzyme, CDK2-7, was efficiently activated in cyclin A-dependent fashion by CDK7 but not at all by CDK2. CDK2-7, moreover, phosphorylated wild-type CDK7 but not CDK2. Our results suggest that the primary amino acid sequence of the T loop plays only a minor role, if any, in determining the specificity of cyclin-dependent CAKs for their CDK substrates and that protein-protein interactions involving sequences outside the T loop can influence substrate specificity both positively and negatively.     

Cell cycle G2/M arrest and activation of cyclin-dependent kinases associated with low-dose paclitaxel-induced sub-G1 apoptosis

Paclitaxel is a potential anti-cancer agent for several malignancies including ovary, breast, and head and neck cancers. This study investigated the kinetics of paclitaxel-induced cell cycle perturbation in two human nasopharyngeal carcinoma (NPC) cell lines, NPC-TW01 and NPC-TW04. NPC cells treated with higher concentrations (0.1 or 1 μM) of paclitaxel showed obvious G2/M arrest and then converted to a cell population with reduced DNA content, which was detected as a sub-G2 peak in the flow cytometric histographs. If a low concentration (5 nM) of paclitaxel was used instead, transient G2/M arrest was observed in NPC cells, which subsequently converted to a sub-G1 form during the treatment period. Internucleosomal fragmentation and chromatin condensation were detectable in these sub-G1 and sub-G2 cells, suggesting that persistent or transient G2/M arrest is a prerequisite step for apoptosis elicited by varying doses of paclitaxel. The levels of cyclins A, B1, D1, E, CDK 1 (CDC 2), CDK 2 and proliferating cell nuclear antigen (PCNA) were unchanged in NPC cells following treatment with any concentration of paclitaxel; however, apoptosis-related cyclin B1-associated CDC 2 kinase was highly activated by paclitaxel even at concentrations as low as 5 nM, which is consistent with the finding that low-dose paclitaxel is also able to induce apoptosis in NPC cells. Activation of cyclin B1-associated CDC 2 kinase seems to be an important G2/M event required for paclitaxel-induced apoptosis, and this activation of cyclin B1/CDC 2 kinase could be attributed to the increased activity of CDK 7 kinase. This revised version was published online in November 2006 with corrections to the Cover Date.     

Phosphorylation of p21 in G2/M promotes cyclin B-Cdc2 kinase activity

Little is known about the posttranslational control of the cyclin-dependent protein kinase (CDK) inhibitor p21. We describe here a transient phosphorylation of p21 in the G2/M phase. G2/M-phosphorylated p21 is short-lived relative to hypophosphorylated p21. p21 becomes nuclear during S phase, prior to its phosphorylation by CDK2. S126-phosphorylated cyclin B1 binds to T57-phosphorylated p21. Cdc2 kinase activation is delayed in p21-deficient cells due to delayed association between Cdc2 and cyclin B1. Cyclin B1-Cdc2 kinase activity and G2/M progression in p21-/- cells are restored after reexpression of wild-type but not T57A mutant p21. The cyclin B1 S126A mutant exhibits reduced Cdc2 binding and has low kinase activity. Phosphorylated p21 binds to cyclin B1 when Cdc2 is phosphorylated on Y15 and associates poorly with the complex. Dephosphorylation on Y15 and phosphorylation on T161 promotes Cdc2 binding to the p21-cyclin B1 complex, which becomes activated as a kinase. Thus, hyperphosphorylated p21 activates the Cdc2 kinase in the G2/M transition.