Calmodulin and cyclin D anchoring sites on the Src-suppressed C kinase substrate, SSeCKS

SSeCKS and its human orthologue, Gravin, are large scaffolding proteins that are thought to facilitate mitogenic control by anchoring key signal mediators such as protein kinase (PK) C, PKA, the plasma membrane associated isoform of alpha-1,4-galactosyltransferase (GalTase), beta2-adrenergic receptor, and cyclins. SSeCKS is also a major PKC substrate and phosphatidylserine-dependent PKC binding protein whose phosphorylation sites shares homology with a site in the MARCKS protein that encodes phosphorylation-sensitive calmodulin (CaM) binding activity. In the present study, we mapped the in vitro binding sites for CaM and cyclins on SSeCKS. Four CaM binding sites were identified by binding assays that conform to the so-called 1-5-10 motif. Notably, CaM binding was antagonized by prephosphorylation of SSeCKS by PKC. We also identified two major cyclin binding (CY) sites that overlap a major PKC phosphorylation site in SSeCKS (Ser(507/515)), and showed that cyclin D binding is attenuated if SSeCKS is prephosphorylated by PKC. These data suggest that the scaffolding activities of SSeCKS are modulated by mitogenically stimulated kinases such as PKC.     

SSeCKS/Gravin/AKAP12 Inhibits Cancer Cell Invasiveness and Chemotaxis by Suppressing a Protein Kinase C- Raf/MEK/ERK Pathway

SSeCKS/Gravin/AKAP12 (“SSeCKS”) encodes a cytoskeletal protein that regulates G₁ → S progression by scaffolding cyclins, protein kinase C (PKC) and PKA. SSeCKS is down-regulated in many tumor types including prostate, and when re-expressed in MAT-LyLu (MLL) prostate cancer cells, SSeCKS selectively inhibits metastasis by suppressing neovascularization at distal sites, correlating with its ability to down-regulate proangiogenic genes including Vegfa. However, the forced re-expression of VEGF only rescues partial lung metastasis formation. Here, we show that SSeCKS potently inhibits chemotaxis and Matrigel invasion, motility parameters contributing to metastasis formation. SSeCKS suppressed serum-induced activation of the Raf/MEK/ERK pathway, resulting in down-regulation of matrix metalloproteinase-2 expression. In contrast, SSeCKS had no effect on serum-induced phosphorylation of the Src substrate, Shc, in agreement with our previous data that SSeCKS does not inhibit Src kinase activity in cells. Invasiveness and chemotaxis could be restored by the forced expression of constitutively active MEK1, MEK2, ERK1, or PKCα. SSeCKS suppressed phorbol ester-induced ERK1/2 activity only if it encoded its PKC binding domain (amino acids 553–900), suggesting that SSeCKS attenuates ERK activation through a direct scaffolding of conventional and/or novel PKC isozymes. Finally, control of MLL invasiveness by SSeCKS is influenced by the actin cytoskeleton: the ability of SSeCKS to inhibit podosome formation is unaffected by cytochalasin D or jasplakinolide, whereas its ability to inhibit MEK1/2 and ERK1/2 activation is nullified by jasplakinolide. Our findings suggest that SSeCKS suppresses metastatic motility by disengaging activated Src and then inhibiting the PKC-Raf/MEK/ERK pathways controlling matrix metalloproteinase-2 expression and podosome formation.     

Cigarette smoke extract induced rat pulmonary artery smooth muscle cells proliferation via PKCα-mediated cyclin D1 expression

Cigarette smoke could induce pulmonary smooth muscle cells (PASMCs) proliferation. Although our previous study had implied the involvement of protein kinase Cα (PKCα), the molecular mechanism underlying PKCα pathway in this process is still unknown. In this study, rat PASMCs were stimulated by cigarette smoke extract (CSE) or PMA (a special activator to PKCα). Two percent CSE and PMA significantly enhanced cyclin D1 expression and cells proliferation. But cyclin D1-specific siRNA successfully inhibited DNA synthesis in CSE-treated or PMA-treated cells. On the other hand, PKCα-specific siRNA significantly suppressed cyclin D1 expression in CSE-treated cells. Moreover, PKCα-specific siRNA resulted in a cell-cycle arrest in G0/G1 and decreased cells number significantly. We conclude that CSE induced rat PASMCs proliferation at least partly via PKCα-mediated cyclin D1 expression.     

A pure estrogen antagonist inhibits cyclin E-Cdk2 activity in MCF-7 breast cancer cells and induces accumulation of p130-E2F4 complexes characteristic of quiescence

Estrogen antagonists inhibit cell cycle progression in estrogen-responsive cells, but the molecular mechanisms are not fully defined. Antiestrogen-mediated G(0)/G(1) arrest is associated with decreased cyclin D1 gene expression, inactivation of cyclin D1-cyclin dependent kinase (Cdk) 4 complexes, and decreased phosphorylation of the retinoblastoma protein (pRb). We now show that treatment of MCF-7 breast cancer cells with the pure estrogen antagonist ICI 182780 results in inhibition of cyclin E-Cdk2 activity prior to a decrease in the G(1) to S phase transition. This decrease was dependent on p21(WAF1/Cip1) since treatment with antisense oligonucleotides to p21 attenuated the effect. Recruitment of p21 to cyclin E-Cdk2 complexes was in turn dependent on decreased cyclin D1 expression since it was apparent following treatment with antisense cyclin D1 oligonucleotides. To define where within the G(0) to S phase continuum antiestrogen-treated cells arrested, we assessed the relative abundance and phosphorylation state of pocket protein-E2F complexes. While both pRb and p107 levels were significantly decreased, p130 was increased 4-fold and was accompanied by the formation of p130.E2F4 complexes and the accumulation of hyperphophorylated E2F4, putative markers of cellular quiescence. Thus, ICI 182780 inhibits both cyclin D1-Cdk4 and cyclin E-Cdk2 activity, resulting in the arrest of MCF-7 cells in a state with characteristics of quiescence (G(0)), as opposed to G(1) arrest.     

The protein kinase C inhibitor enzastaurin exhibits antitumor activity against uveal melanoma

GNAQ mutations at codon 209 have been recently identified in approximately 50% of uveal melanomas (UM) and are reported to be oncogenic through activating the MAPK/Erk1/2 pathway. Protein kinase C (PKC) is a component of signaling from GNAQ to Erk1/2. Inhibition of PKC might regulate GNAQ mutation-induced Erk1/2 activation, resulting in growth inhibition of UM cells carrying GNAQ mutations. UM cells carrying wild type or mutant GNAQ were treated with the PKC inhibitor enzastaurin. Effects on proliferation, apoptosis, and signaling events were evaluated. Enzastaurin downregulated the expression of several PKC isoforms including PKCβII PKCθ, PKCε and/or their phosphorylation in GNAQ mutated cells. Downregulation of these PKC isoforms in GNAQ mutated cells by shRNA resulted in reduced viability. Enzastaurin exhibited greater antiproliferative effect on GNAQ mutant cells than wild type cells through induction of G1 arrest and apoptosis. Enzastaurin-induced G1 arrest was associated with inhibition of Erk1/2 phosphorylation, downregulation of cyclin D1, and accumulation of cyclin dependent kinase inhibitor p27(Kip1). Furthermore, enzastaurin reduced the expression of antiapoptotic Bcl-2 and survivin in GNAQ mutant cells. Inhibition of Erk1/2 phosphorylation with a MEK specific inhibitor enhanced the sensitivity of GNAQ wild type cells to enzastaurin, accompanied by p27(Kip1) accumulation and/or inhibition of enzastaurin-induced survivin and Bcl-2 upregulation. PKC inhibitors such as enzastaurin have activity against UM cells carrying GNAQ mutations through inhibition of the PKC/Erk1/2 pathway and induction of G1 arrest and apoptosis. Inhibition of the PKC pathway provides a basis for clinical investigation in patients with UM.     

Troglitazone inhibits growth of MCF-7 breast carcinoma cells by targeting G1 cell cycle regulators

Peroxisome proliferator activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily. Ligand activation of PPARgamma has been shown to cause growth arrest in several human tumor cell types, but the underlying molecular mechanism has not been elucidated. We report here that the PPARgamma ligand troglitazone (TRO) inhibited MCF-7 cell proliferation by blocking events critical for G1 --> S progression. Flow cytometry demonstrated that TRO at 20 microM increased the percentage of cells in G1 from 51 to 69% after 24 h. Accumulation of cells in G1 was accompanied by an attenuation of Rb protein phosphorylation associated with decreased CDK4 and CDK2 activities. Inhibition of CDK activity by TRO correlates with decreased protein levels for several G1 regulators of Rb phosphorylation (cyclin D1, and CDKs 2, 4, and 6). Overexpression of cyclin D1 partially rescued MCF-7 cells from TRO-mediated G1 arrest. Targeting of G1 regulatory proteins, particularly cyclin D1, and the resulting induction of G1 arrest by TRO may provide a novel antiproliferative therapy for human breast cancer.     

Altered p27(Kip1) phosphorylation, localization, and function in human epithelial cells resistant to transforming growth factor beta-mediated G(1) arrest

p27(Kip1) is an important effector of G(1) arrest by transforming growth factor beta (TGF-beta). Investigations in a human mammary epithelial cell (HMEC) model, including cells that are sensitive (184(S)) and resistant (184A1L5(R)) to G(1) arrest by TGF-beta, revealed aberrant p27 regulation in the resistant cells. Cyclin E1-cyclin-dependent kinase 2 (cdk2) and cyclin A-cdk2 activities were increased, and p27-associated kinase activity was detected in 184A1L5(R) cells. p27 from 184A1L5(R) cells was localized to both nucleus and cytoplasm, showed an altered profile of phosphoisoforms, and had a reduced ability to bind and inhibit cyclin E1-cdk2 in vitro when compared to p27 from the sensitive 184(S) cells. In proliferating 184A1L5(R) cells, more p27 was associated with cyclin D1-cdk4 complexes than in 184(S). While TGF-beta inhibited the formation of cyclin D1-cdk4-p27 complexes in 184(S) cells, it did not inhibit the assembly of cyclin D1-cdk4-p27 complexes in the resistant 184A1L5(R) cells. p27 phosphorylation changed during cell cycle progression, with cyclin E1-bound p27 in G(0) showing a different phosphorylation pattern from that of cyclin D1-bound p27 in mid-G(1). These data suggest a model in which TGF-beta modulates p27 phosphorylation from its cyclin D1-bound assembly phosphoform to an alternate form that binds tightly to inhibit cyclin E1-cdk2. Altered phosphorylation of p27 in the resistant 184A1L5(R) cells may favor the binding of p27 to cyclin D1-cdk4 and prevent its accumulation in cyclin E1-cdk2 in response to TGF-beta.     

Phorbol ester-induced G1 phase arrest selectively mediated by protein kinase Cdelta-dependent induction of p21

Although protein kinase C (PKC) has been widely implicated in the positive and negative control of proliferation, the underlying cell cycle mechanisms regulated by individual PKC isozymes are only partially understood. In this report, we show that PKCdelta mediates phorbol ester-induced G1 arrest in lung adenocarcinoma cells and establish an essential role for this novel PKC in controlling the expression of the cell cycle inhibitor p21. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) in early G1 phase impaired progression of lung adenocarcinoma cells into S phase, an effect that was completely abolished by specific depletion of PKCdelta, but not PKCalpha. Although the PKC effect was unrelated to the inhibition of cyclin D1 expression, PKC activation significantly up-regulated p21 and down-regulated Rb hyperphosphorylation and cyclin A expression. Elevations in p21 mRNA and protein by PMA were mediated by PKCdelta but not PKCalpha. Studies using luciferase reporters also revealed an essential role for PKCdelta in the PMA-induced inhibition of Rb-dependent cyclin A promoter activity. Finally, we showed that the cell cycle inhibitory effect of PKCdelta is greatly attenuated by RNA interference-mediated knock-down of p21. Our results identify a novel link between PKCdelta and G1 arrest via p21 up-regulation and highlight the complexities in the downstream effectors of PKC isozymes in the context of cell cycle progression and proliferation.     

ERK1/2 and p38 cooperate to delay progression through G1 by promoting cyclin D1 protein turnover

The conditional kinase DeltaMEKK3:ER allows activation of JNK, p38 and ERK1/2 without overt cellular stress or damage and has proved useful in understanding how these pathways regulate apoptosis and cell cycle progression. We have previously shown that activation of DeltaMEKK3:ER causes a sustained G(1) cell cycle arrest which requires p21(CIP1), with ERK1/2 and p38 cooperating to promote p21(CIP1) expression. In cells lacking p21(CIP1), DeltaMEKK3:ER causes only a transient delay in cell cycle re-entry. We now show that this delay in cell cycle re-entry is due to a reduction in cyclin D1 levels. Activation of DeltaMEKK3:ER promotes the proteasome-dependent turnover of cyclin D1; this requires phosphorylation of threonine 286 (T(286)) and expression of cyclin D1T(286)A rescues the delay in G(1)/S progression. DeltaMEKK3:ER-dependent phosphorylation of T(286) does not appear to be mediated by GSK3beta but requires activation of the ERK1/2 and p38 pathways. ERK1/2 can physically associate with cyclin D1 but activation of ERK1/2 alone is not sufficient for phosphorylation of T(286). Rather, cyclin D1 phosphorylation appears to require coincident activation of ERK1/2 and p38. Thus activation of DeltaMEKK3:ER promotes a sustained G(1) cell cycle arrest by a bipartite mechanism involving the rapid destruction of cyclin D1 and the slower more prolonged expression of p21(CIP1). This has parallels with the bipartite response to ionizing radiation and p53-independent mechanisms of G(1) cell cycle arrest in simple organisms such as yeast.     

Dna damage-induced G(1) arrest in hematopoietic cells is overridden following phosphatidylinositol 3-kinase-dependent activation of cyclin-dependent kinase 2

Exposure of hematopoietic cells to DNA-damaging agents induces p53-independent cell cycle arrest at a G(1) checkpoint. Previously, we have shown that this growth arrest can be overridden by cytokine growth factors, such as erythropoietin or interleukin-3, through activation of a phosphatidylinositol 3-kinase (PI 3-kinase)/Akt-dependent signaling pathway. Here, we show that gamma-irradiated murine myeloid 32D cells arrest in G(1) with active cyclin D-cyclin-dependent kinase 4 (Cdk4) but with inactive cyclin E-Cdk2 kinases. The arrest was associated with elevated levels of the Cdk inhibitors p21(Cip1) and p27(Kip1), yet neither was associated with Cdk2. Instead, irradiation-induced inhibition of cyclin E-Cdk2 correlated with absence of the activating threonine-160 phosphorylation on Cdk2. Cytokine treatment of irradiated cells induced Cdk2 phosphorylation and activation, and cells entered into S phase despite sustained high-level expression of p21 and p27. Notably, the PI 3-kinase inhibitor, LY294002, completely blocked cytokine-induced Cdk2 activation and cell growth in irradiated 32D cells but not in nonirradiated cells. Together, these findings demonstrate a novel mechanism underlying the DNA damage-induced G(1) arrest of hematopoietic cells, that is, inhibition of Cdk2 phosphorylation and activation. These observations link PI 3-kinase signaling pathways with the regulation of Cdk2 activity.     

Inhibition of G1 cyclin-dependent kinase activity during growth arrest of human astrocytoma cells by the pyrrolo-1,5-benzoxazepine,PBOX-21

The present study examines the molecular mechanisms by which a member of a novel series of pyrrolo-1,5-benzoxazepines, PBOX-21, induces G1 arrest in 1321N1 cells. PBOX-21-induced G1 arrest is preceded by both a decrease in CDK2 kinase activity, which is critical for the G1/S transition, and a downregulation in cyclin D(3) protein expression levels, suggesting that these two events may be crucially involved in the mediation of the cell cycle arrest. The decrease in CDK2 activity may be due to an observed decrease in CDK2 protein levels following PBOX-21 treatment. Coinciding with the arrest is a reduction in the activity of CDK4, due to either the observed PBOX-21 induced downregulation in CDK4 expression, or a reduction in complex formation between cyclin D(3)-CDK4 leading to a decrease in the levels of active cyclin D(3)-CDK4 complexes with kinase activity. The level of CDK6 activity was also seen to be reduced following PBOX-21 treatment, also possibly due to a reduction in complex formation with cyclin D(3). However, this reduction in CDK6 kinase activity was not seen until after PBOX-21-induced G1 arrest has reached its maximum, and therefore may be viewed as a consequence of, and a method of maintaining the PBOX-21-induced arrest, rather than a cause. Also in parallel with the G1 arrest elicited by PBOX-21 is an upregulation in the universal CDK inhibitor, p21. Furthermore, the retinoblastoma protein (Rb), a substrate of CDK2 and CDK6, whose phosphorylation is necessary for cell cycle progression, becomes hypophosphorylated. These results indicate that PBOX-21 exerts its growth inhibitory effects through the modulation of the expression and activity of several key G1 regulatory proteins.     

Overexpression of the signaling adapter FRS2 reconstitutes the cell cycle deficit of a nerve growth factor non-responsive TrkA receptor mutant

We have characterized the cell cycle deficit of a novel TrkA receptor mutant (TrkAS3) that fails to support nerve growth factor (NGF)-dependent cell cycle arrest and neurite outgrowth. TrkAS3 receptors fail to support an NGF-dependent increase in the expression of cyclin D1 and the cell cycle inhibitor, p21(Waf1/Cip1), two important regulators of G(1) /S transition, and do not down-regulate expression of the G(2) /M phase marker, cdc2/cdk1, or the S phase marker, proliferating cell nuclear antigen. Moreover, NGF-activated TrkAS3 receptors do not down-regulate cyclin-dependent kinase 4 phosphorylation of the retinoblastoma protein, essential for G(1) arrest, in comparison to NGF-activated wild-type TrkA. Collectively these data indicate that TrkAS3 receptors fail to support NGF-dependent G(1) arrest. Interestingly, ectopic expression of regulators of G(1) /S arrest, such as cyclin D1 or inhibitors of cell cycle (p21(Waf1/Cip1), p16(INK4A) ), or the fibroblast growth factor (FGF) receptor substrate-2 (FRS2) in cells expressing TrkAS3 reconstitutes NGF-dependent neurite outgrowth. Collectively, these data suggest a model in which NGF-stimulated TrkA-dependent activation of FRS2 supports neurite outgrowth through a mechanism that likely involves the induction of p21(Waf1/Cip1) expression and the arrest of cells at G(1) /S.     

Fbxl12 triggers G1 arrest by mediating degradation of calmodulin kinase I

Cell cycle progression through its regulatory control by changes in intracellular Ca^2 + levels at the G1/S transition mediates cellular proliferation and viability. Ca^2 +/CaM-dependent kinase 1 (CaMKI) appears critical in regulating the assembly of the cyclin D1/cdk4 complex essential for G1 progression, but how this occurs is unknown. Cyclin D1/cdk4 assembly in the early G1 phase is also regulated via binding to p27. Here, we show that a ubiquitin E3 ligase component, F-box protein Fbxl12, mediates CaMKI degradation via a proteasome-directed pathway leading to disruption of cyclin D1/cdk4 complex assembly and resultant G1 arrest in lung epithelia. We also demonstrate that i) CaMKI phosphorylates p27 at Thr¹⁵⁷ and Thr¹⁹⁸ in human cells and at Thr¹⁷⁰ and Thr¹⁹⁷ in mouse cells to modulate its subcellular localization; ii) Fbxl12-induced CaMKI degradation attenuates p27 phosphorylation at these sites in early G1 and iii) activation of CaMKI during G1 transition followed by p27 phosphorylation appears to be upstream to other p27 phosphorylation events, an effect abrogated by Fbxl12 overexpression. Lastly, known inducers of G1 arrest significantly increase Fbxl12 levels in cells. Thus, Fbxl12 may be a previously uncharacterized, functional growth inhibitor regulating cell cycle progression that might be used for mechanism-based therapy.     

Control of protein kinase C activity, phorbol ester-induced cytoskeletal remodeling, and cell survival signals by the scaffolding protein SSeCKS/GRAVIN/AKAP12

The product of the SSeCKS/GRAVIN/AKAP12 gene ("SSeCKS") is a major protein kinase (PK) C substrate that exhibits tumor- and metastasis-suppressing activity likely through its ability to scaffold multiple signaling mediators such as PKC, PKA, cyclins, calmodulin, and Src. Although SSeCKS and PKCα bind phosphatidylserine, we demonstrate that phosphatidylserine-independent binding of PKC by SSeCKS is facilitated by two homologous SSeCKS motifs, EG(I/V)(T/S)XWXSFK(K/R)(M/L)VTP(K/R)K(K/R)X(K/R)XXXEXXXE(E/D) (amino acids 592-620 and 741-769). SSeCKS binding to PKCα decreased kinase activity and was dependent on the two PKC-binding motifs. SSeCKS scaffolding of PKC was increased in confluent cell cultures, correlating with significantly increased SSeCKS protein levels and decreased PKCα activity, suggesting a role for SSeCKS in suppressing PKC activation during contact inhibition. SSeCKS-null mouse embryo fibroblasts displayed increased relative basal and phorbol ester (phorbol 12-myristate 13-acetate)-induced PKC activity but were defective in phorbol 12-myristate 13-acetate-induced actin cytoskeletal reorganization and cell shape change; these responses could be rescued by the forced expression of full-length SSeCKS but not by an SSeCKS variant deleted of its PKC-binding domains. Finally, the PKC binding sites in SSeCKS were required to restore cell rounding and/or decreased apoptosis in phorbol ester-treated LNCaP, LNCaP-C4-2, and MAT-LyLu prostate cancer cells. Thus, PKC-mediated remodeling of the actin cytoskeleton is likely regulated by the ability of SSeCKS to control PKC signaling and activity through a direct scaffolding function.     

Roles of histone acetylation modification in basal and inducible expression of hsp26 gene in D. melanogaster

Src-suppressed C kinase substrate (SSeCKS) plays a role in membrane-cytoskeletal remodeling to regulate mitogenesis, cell differentiation, and motility. Previous study showed that lipopolysaccharide (LPS) induced a selective and strong expression of SSeCKS in the vascular endothelial cells of lung. Here we show that LPS stimulation elevated expression of SSeCKS mRNA and protein in Rat pulmonary microvascular endothelial cell (RPMVEC). LPS potentiated SSeCKS phosphorylation in a time- and dose-dependent manner, and partly induced translocation of SSeCKS from the cytosol to the membrane after LPS challenge. The PKC inhibitor, Calphostin C, significantly decreased LPS-induced phosphorylation of SSeCKS, inhibited SSeCKS translocation and actin cytoskeleton reorganization after LPS challenge, suggesting that PKC may play a role in LPS-induced SSeCKS translocation and actin rearrangement. We conclude that SSeCKS is located downstream of PKC and that SSeCKS and PKC are both necessary for LPS-induced stress fiber formation. Chun Cheng and Haiou Liu are contributed equally to this work.     

Dysregulated expression of cyclin D1 in normal human mammary epithelial cells inhibits all-trans-retinoic acid-mediated G0/G1-phase arrest and differentiation in vitro.

Overexpression of cyclin D1 protein is observed in the majority of breast cancers, suggesting that dysregulated expression of cyclin D1 might be a critical event in breast cancer carcinogenesis. We investigated whether retroviral-mediated expression of cyclin D1 might affect all-trans-retinoic acid (ATRA)-mediated growth inhibition and differentiation of normal cultured human mammary epithelial cells (HMECs). HMECs treated with 1.0 microM ATRA undergo irreversible growth inhibition starting at 24 h and complete G0/G1-phase arrest by Day 3. Cyclin D1 protein levels are observed to decrease in association with the initiation of growth arrest starting at 24 h and then increase by approximately 35% on Day 3. Concomitant with this observed increase in cyclin D1, HMECs undergo morphologic changes consistent with progression to a more differentiated phenotype, including an increase in cell size, increased cell spreading, increased tonofilaments, and accumulation of cytoplasmic vesicles containing lipid. Dysregulated expression of cyclin D1 in HMECs results in inhibition of G0/G1-phase arrest mediated by ATRA. In addition, HMECs expressing exogenous cyclin D1 are resistant to differentiation by ATRA. Our results suggest that coordinated expression of cyclin D1 may be critical for normal mammary epithelial cell homeostasis, and dysregulated expression of cyclin D1 might result in retinoid resistance and promote mammary carcinogenesis.     

L-leucine increases [3H]-thymidine incorporation in chicken hepatocytes: involvement of the PKC, PI3K/Akt, ERK1/2, and mTOR signaling pathways

This study examined how L-leucine affected DNA synthesis and cell cycle regulatory protein expression in cultured primary chicken hepatocytes. L-Leucine promoted DNA synthesis in a dose- and time-dependent manner, with concomitant increases in cyclin D1 and cyclin E expression. Phospholipase C (PLC) and protein kinase C (PKC) mediated the L-leucine-induced increases in [3H]-thymidine incorporation and cyclin D1/CDK4 and cyclin E/CDK2 expression, as U73122 (a PLC inhibitor) or bisindolylmaleimide I (a PKC blocker) inhibited these effects. L-Leucine also increased PKC phosphorylation and intracellular Ca2+ levels. L-Leucine-mediated increases in [3H]-thymidine incorporation and cyclin/CDK expression were sensitive to LY 294002 (PI3K inhibitor), Akt inhibitor, PD 98059 (MEK inhibitor). It was also observed that L-leucine-induced increases of cyclin/CDK expression were inhibited by PI3K siRNA and ERK siRNA; L-leucine increased extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt phosphorylation levels. Bisindolylmaleimide I attenuated L-leucine-induced phosphorylation of ERK1/2 but did not influence Akt phosphorylation, and PI3K siRNA and LY 294002 inhibited L-leucine-induced ERK1/2 phosphorylation, suggesting some cross-talk between the PKC and ERK1/2 or PI3K/Akt and ERK1/2 pathways. L-Leucine also increased the levels of phosphorylated molecular target of rapamycin (mTOR) and two of its targets, ribosomal protein S6 kinase (p70S6K), and 4E binding protein 1 (4E-BP1); furthermore, rapamycin (an mTOR inhibitor) blocked all of the mitogenic effects of L-leucine. In addition, Akt inhibitor blocked L-leucine-induced mTOR phosphorylation. In conclusion, L-leucine stimulated DNA synthesis and promoted cell cycle progression in primary cultured chicken hepatocytes through PKC, ERK1/2, PI3K/Akt, and mTOR.     

Mirk/dyrk1B kinase destabilizes cyclin D1 by phosphorylation at threonine 288

The phosphorylation of cyclin D1 at threonine 286 by glycogen synthase kinase 3beta (GSK3beta) has been shown to be required for the ubiquitination and nuclear export of cyclin D1 and its subsequent degradation in the proteasome. The mutation of the nearby residue, threonine 288, to nonphosphorylatable alanine has also been shown to reduce the ubiquitination of cyclin D1, suggesting that phosphorylation at threonine 288 may also lead to degradation of cyclin D1. We now demonstrate that the G(0)/G(1)-active arginine-directed protein kinase Mirk/dyrk1B binds to cyclin D1 and phosphorylates cyclin D1 at threonine 288 in vivo and that the cyclin D1-T288A construct is more stable than wild-type cyclin D1. Transient overexpression of Mirk in nontransformed Mv1Lu lung epithelial cells blocked cells in G(0)/G(1). Depletion of endogenous Mirk by RNA interference increased cyclin D1 protein levels but not mRNA levels, indicating that Mirk destabilizes cyclin D1 protein. Destabilization was confirmed by induction of a stable Mirk transfectant of Mv1Lu cells, which blocked cell migration (Zou, Y., Lim, S., Lee, K., Deng, X., and Friedman, E. (2003) J. Biol. Chem. 278, 49573-49581), and caused a decrease in the half-life of endogenous cyclin D1, concomitant with an increase in Mirk expression. In vitro cyclin D1 was phosphorylated in an additive fashion by Mirk and GSK3beta. Mirk-phosphorylated cyclin D1 mutated at the GSK3beta phosphorylation site and was capable of phosphorylating cyclin D1 in the presence of the GSK3beta inhibitor LiCl. Mirk may function together with GSK3beta to assist cell arrest in G(0)/G(1) by destabilizing cyclin D1.