Xiaobing Deng

The Cyclin-dependent Kinase Inhibitor p27Kip1 Is Stabilized in G0 by Mirk/dyrk1B Kinase

Elevated levels of the cyclin-dependent kinase (CDK) inhibitor p27 block the cell in G0/G1 until mitogenic signals activate G1 cyclins and initiate proliferation. Post-translational regulation of p27 by different phosphorylation events is critical in allowing cells to proceed through the cell cycle. We now demonstrate that the arginine-directed kinase, Mirk/dyrk1B, is maximally active in G0 in NIH3T3 cells, when it stabilizes p27 by phosphorylating it at Ser-10. The phospho-mimetic mutant p27-S10D was more stable, and the non-phosphorylatable mutant p27-S10A was less stable than wild-type when expressed in G0-arrested cells. Following phosphorylation by Mirk, p27 remains a functional CDK inhibitor, capable of binding to CDK2. Mirk did not induce the translocation of p27 from the nucleus in G0, but instead co-localized with nuclear p27. Depletion of Mirk by RNA interference decreased the phosphorylation of p27 at Ser-10 and the stability of endogenous p27. RNAi to Mirk increased cell entry from G0 into G1 as shown by increased expression of proliferating cell nuclear antigen and decreased expression of p27. These data suggest a model in which Mirk increases the amount of nuclear p27 by stabilizing it during G0 when Mirk is most abundant. Mitogen stimulation then causes cells to enter G1, reduces Mirk levels (Deng, X., Ewton, D., Pawlikowski, B., Maimone, M., and Friedman, E. (2003) J. Biol. Chem. 278, 41347-41354), and initiates the translocation of p27 to the cytoplasm. In addition, depletion of Mirk by RNAi in postmitotic C2C12 myoblasts decreased protein but not mRNA levels of p27, suggesting that stabilization of p27 by Mirk also occurs during differentiation.

Mirk/dyrk1B is a Rho-induced kinase active in skeletal muscle differentiation.

The Rho family of small GTPases regulates numerous signaling pathways that control the organization of the cytoskeleton, transcription factor activity, and many aspects of the differentiation of skeletal myoblasts. We now demonstrate that the kinase Mirk (minibrain-related kinase)/dyrk1B is induced by members of the Rho-family in myoblasts and that Mirk is active in skeletal muscle differentiation. Mirk is an arginine-directed serine/threonine kinase which is expressed at elevated levels in skeletal muscle compared with other normal tissues. A Mirk promoter construct was activated when C2C12 myoblasts were switched from growth to differentiation medium and was also activated by the Rho family members RhoA, Cdc42, and to a lesser degree Rac1, but not by MyoD or Myf5. Mirk protein levels increased following transient expression of constitutively active Cdc42-QL, RhoA-QL, or Rac1-QL in C2C12 cells. High concentrations of serum mitogens down-regulated Mirk through activation of the Ras-MEK-Erk pathway. As a result, Mirk transcription was induced by the MEK1 inhibitor PD98059 and by the switch from growth to differentiation medium. Mirk was induced with similar kinetics to another Rho-induced differentiation gene, myogenin. Mirk protein levels increased 10-fold within 24–48 h after primary cultured muscle cells; C2C12 mouse myoblasts or L6 rat myoblasts were induced to differentiate. Thus Mirk was induced following the commitment stage of myogenesis. Stable overexpression of Mirk enabled myoblasts to fuse more rapidly when placed in differentiation medium. The function of Mirk in muscle differentiation was established by depletion of endogenous Mirk by small interfering RNA, which prevented myoblast fusion into myotubes and inhibited induction of markers of differentiation, including myogenin, fast twitch troponin T, and muscle myosin heavy chain. Other members of the dyrk/minibrain/HIPK family of kinases in lower organisms have been shown to regulate the transition from growth to differentiation, and Mirk is now shown to participate in skeletal muscle development.

The CDK Inhibitor p27kip1 is stabilized in G0 by Mirk/dyrk1B kinase

Elevated levels of the cyclin-dependent kinase (CDK) inhibitor p27 block the cell in G0/G1 until mitogenic signals activate G1 cyclins and initiate proliferation. Post-translational regulation of p27 by different phosphorylation events is critical in allowing cells to proceed through the cell cycle. We now demonstrate that the arginine-directed kinase, Mirk/dyrk1B, is maximally active in G0 in NIH3T3 cells, when it stabilizes p27 by phosphorylating it at Ser-10. The phospho-mimetic mutant p27-S10D was more stable, and the non-phosphorylatable mutant p27-S10A was less stable than wild-type when expressed in G0-arrested cells. Following phosphorylation by Mirk, p27 remains a functional CDK inhibitor, capable of binding to CDK2. Mirk did not induce the translocation of p27 from the nucleus in G0, but instead co-localized with nuclear p27. Depletion of Mirk by RNA interference decreased the phosphorylation of p27 at Ser-10 and the stability of endogenous p27. RNAi to Mirk increased cell entry from G0 into G1 as shown by increased expression of proliferating cell nuclear antigen and decreased expression of p27. These data suggest a model in which Mirk increases the amount of nuclear p27 by stabilizing it during G0 when Mirk is most abundant. Mitogen stimulation then causes cells to enter G1, reduces Mirk levels (Deng, X., Ewton, D., Pawlikowski, B., Maimone, M., and Friedman, E. (2003) J. Biol. Chem. 278, 41347-41354), and initiates the translocation of p27 to the cytoplasm. In addition, depletion of Mirk by RNAi in postmitotic C2C12 myoblasts decreased protein but not mRNA levels of p27, suggesting that stabilization of p27 by Mirk also occurs during differentiation.

The Kinase Mirk/Dyrk1B Mediates Cell Survival in Pancreatic Ductal Adenocarcinoma

Ductal adenocarcinoma of the pancreas is almost uniformly lethal as this cancer is invariably detected at an advanced stage and is resistant to treatment. The serine/threonine kinase Mirk/Dyrk1B has been shown to be antiapoptotic in rhabdomyosarcomas. We have now investigated whether Mirk might mediate survival in another cancer in which Mirk is widely expressed, pancreatic ductal adenocarcinoma. Mirk was an active kinase in each pancreatic cancer cell line where it was detected. Mirk knockdown by RNA interference (RNAi) reduced the clonogenicity of Panc1 pancreatic cancer cells 4-fold and decreased tumor cell number, showing that Mirk mediates survival in these cells. Mirk knockdown by synthetic duplex RNAis in Panc1, AsPc1, and SU86.86 pancreatic cancer cells induced apoptosis and enhanced the apoptosis induced by gemcitibine. Mirk knockdown did not increase the abundance or activation of Akt. However, four of five pancreatic carcinoma cell lines exhibited either elevated Mirk activity or elevated Akt activity, suggesting that pancreatic cancer cells primarily rely on Mirk or Akt for survival signaling. Mirk protein was detected by immunohistochemistry in 25 of 28 cases (89%) of pancreatic ductal adenocarcinoma, with elevated expression in 11 cases (39%). Increased expression of Mirk was seen in pancreatic carcinomas compared with primary cultures of normal ductal epithelium by serial analysis of gene expression and by immunohistochemistry. Thus, Mirk is a survival factor for pancreatic ductal adenocarcinoma. Because knockout of Mirk does not cause embryonic lethality, Mirk is not essential for normal cell growth and may represent a novel therapeutic target. (Cancer Res 2006; 66(8): 4149-58)

Oncogenic c-Ki-ras but Not Oncogenic c-Ha-ras Up-regulates CEA Expression and Disrupts Basolateral Polarity in Colon Epithelial Cells

Colon carcinomas commonly contain mutations in Ki-ras4B, but very rarely in Ha-ras, suggesting that different Ras isoforms may have distinct functions in colon epithelial cell biology. In an earlier study we had demonstrated that oncogenic Ki-ras4BVal-12, but not oncogenic Ha-ras Val-12, blocks the apicobasal polarization of colon epithelial cells by preventing normal glycosylation of the integrin β1 chain of the collagen receptor. As a result, only the Ki-ras mutated cells exhibited altered cell to substratum attachment, whereas mutation of either Ras isoform activated mitogen-activated protein kinases. We have now asked whether intercellular adhesion proteins implicated in establishing basolateral polarity in colon epithelial cells are modulated by oncogenic Ki-Ras4BVal-12 proteins but not oncogenic Ha-RasVal-12 proteins. The embryonic adhesion protein carcinoembryonic antigen (CEA) was up-regulated on the mRNA and protein levels in each of three stable Ki-ras Val-12 transfectant lines but in none of three stable Ha-ras Val-12 transfectant lines. The elevated protein levels of CEA in Ki-ras4BVal-12 transfectant cells were decreased by blocking expression of Ki-ras4BVal-12 with antisense oligonucleotides. N-cadherin levels were decreased in only the Ki-rastransfectants, whereas E-cadherin levels were unchanged. Immunohistochemical analysis demonstrated that Ki-ras4BVal-12 transfectant cells did not polarize into cells with discrete apical and basal regions and so could not restrict expression of CEA to the apical region. These unpolarized cells displayed elevated levels of CEA all along their surface membrane where CEA mediated random, multilayered associations of tumor cells. This aggregation was both calcium-independent and blocked by Fab′ fragments of anti-CEA monoclonal antibody col-1. Trafficking of the lysosomal cysteine protease cathepsin B may also be altered when cell polarity cannot be established. Ki-ras4BVal-12transfectant cells expressed 2-fold elevated protein levels of the lysosomal cysteine protease cathepsin B but did not up-regulate cathepsin B mRNA expression. One function of oncogenic c-Ki-Ras proteins in colon cancer progression may be to up-regulate CEA and thus to prevent the lateral adhesion of adjacent colon epithelial cells that normally form a monolayer in vivo.

Transforming Growth Factor β1 Induces Proliferation in Colon Carcinoma Cells by Ras-dependent, smad-independent Down-regulation of p21cip1

Transforming growth factor β1 (TGFβ1) can act as a tumor suppressor or a tumor promoter depending on the characteristics of the malignant cell. We recently demonstrated that colon carcinoma cells transfected with oncogenic cellularK-rasV12, but not oncogenic cellular H-rasV12, switched from TGFβ1-insensitive to TGFβ1-growth-stimulated and also became more invasive (Yan, Z., Deng, X., and Friedman, E. (2001)J. Biol. Chem. 276, 1555–1563). We now demonstrate that TGFβ1 growth stimulation of colon carcinoma cells is Ras-dependent and smad-independent. In U9 colon carcinoma cells, which are responsive to TGFβ1 by growth stimulation, a truncating mutation at Gln-311 was found in thesmad4 gene. Very little smad4 protein was detected in these cells. Loss of smad4 protein was confirmed by functional studies. In U9 cells co-transfected wild-type smad4, but not mutant smad4, mediated response of the 3TP-lux and pSBE promoter reporter constructs to TGFβ1. Proliferation initiated by TGFβ1 in U9 cells required Ras-mediated down-regulation of p21cip1 protein. Less p21cip1 was associated with cdk2·cyclin complexes in TGFβ1-treated U9 cells, and the cdk2 complexes had increased kinase activity. Elevation of p21cip1 levels diminished proliferative response to TGFβ1. U9 cells expressing DN-N17ras neither proliferated in response to TGFβ1 nor down-regulated the cdk inhibitor p21cip1, and TGFβ1 activation of 3TP-lux in U9 cells was inhibited by DN-N17ras in a dose-dependent manner. TGFβ1 also decreased p21cip1 levels and stimulated proliferation in SW480 cells, which express mutant K-Ras but no smad4 protein. TGFβ1 did not activate or inhibit the p21cip1 promoter construct in U9 cells even in the presence of co-transfected smad4, or alter p21cip1 mRNA levels. Thus the decrease in p21cip1 levels was mediated by a TGFβ-initiated Ras-dependent, but smad-independent post-transcriptional mechanism.

Inactivation of Mirk/Dyrk1B Kinase Targets Quiescent Pancreatic Cancer Cells

A major problem in the treatment of cancer arises from quiescent cancer cells that are relatively insensitive to most chemotherapeutic drugs and radiation. Such residual cancer cells can cause tumor regrowth or recurrence when they reenter the cell cycle. Earlier studies showed that levels of the serine/theronine kinase Mirk/dyrk1B are elevated up to 10-fold in quiescent G0 tumor cells. Mirk uses several mechanisms to block cell cycling, and Mirk increases expression of antioxidant genes that decrease reactive oxygen species (ROS) levels and increase quiescent cell viability. We now show that a novel small molecule Mirk kinase inhibitor blocked tumor cells from undergoing reversible arrest in a quiescent G0 state and enabled some cells to exit quiescence. The inhibitor increased cycling in Panc1, AsPc1, and SW620 cells that expressed Mirk, but not in HCT116 cells that did not. Mirk kinase inhibition elevated ROS levels and DNA damage detected by increased phosphorylation of the histone protein H2AX and by S-phase checkpoints. The Mirk kinase inhibitor increased cleavage of the apoptotic proteins PARP and caspase 3, and increased tumor cell kill several-fold by gemcitabine and cisplatin. A phenocopy of these effects occurred following Mirk depletion, showing drug specificity. In previous studies Mirk knockout or depletion had no detectable effect on normal tissue, suggesting that the Mirk kinase inhibitor could have a selective effect on cancer cells expressing elevated levels of Mirk kinase. Mol Cancer Ther; 10(11); 2104–14. ©2011 AACR.

Rapid turnover of cell-cycle regulators found in Mirk/dyrk1B transfectants.

Mirk/dyrk1B is an arginine‐directed protein kinase, which functions as a transcriptional activator and mediates serum‐free growth of colon carcinoma cells by an unknown mechanism. We now report that turnover of the cdk inhibitor p27kip1 and the G1‐phase cyclin cyclin D1 is enhanced in each of 4 Mirk stable transfectants compared to vector control transfectants and Mirk kinase‐inactive mutant transfectants. This enhanced turnover is proteasome‐dependent and leads to lower protein levels of both p27kip1 and cyclin D1. Lower protein levels of the cdk inhibitor p21cip1 were also observed in the 4 Mirk stable transfectants. Mirk did not alter the activity of a p27kip1 promoter construct or p27kip1 mRNA levels by stable expression, indicating that the decrease in p27kip1 protein levels was due to a posttranscriptional mechanism. These data are consistent with mirk enhancing the expression of some component common to the proteolysis of both p27kip1 and cyclin D1.