Mirk/dyrk1B kinase is upregulated following inhibition of mTOR.

The PI3K/PTEN/Akt/mTOR/p70S6K pathway is one of the most frequently deregulated signaling pathways in solid tumors and has a functional role in drug resistance. However, targeting this pathway leads to compensatory activation of several mediators of cell survival. Expression of the reactive oxygen species-controlling kinase Mirk/dyrk1B was increased severalfold by the mammalian target of rapamycin (mTOR) inhibitors RAD001, WYE354 and rapamycin, with less effect by the Akt inhibitors AZD5363 and MK-2206.

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 G(0) tumor cells.

Mirk regulates the exit of colon cancer cells from quiescence.

Mirk/Dyrk1B is a serine/threonine kinase widely expressed in colon cancers. Serum starvation induced HD6 colon carcinoma cells to enter a quiescent G0 state, characterized by a 2N DNA content and a lower RNA content than G1 cells. Compared with cycling cells, quiescent cells exhibited 16-fold higher levels of the retinoblastoma protein p130/Rb2, which sequesters E2F4 to block entry into G1, 10-fold elevated levels of the CDK inhibitor p27kip1, and 10-fold higher levels of Mirk.

Mirk/Dyrk1B maintains the viability of quiescent pancreatic cancer cells by reducing levels of reactive oxygen species.

The kinase Mirk/dyrk1B mediated the clonogenic growth of pancreatic cancer cells in earlier studies. It is now shown that Mirk levels increased 7-fold in SU86.86 pancreatic cancer cells when over a third of the cells were accumulated in a quiescent G(0) state, defined by Hoechst/Pyronin Y staining.

The survival kinase Mirk/Dyrk1B is a downstream effector of oncogenic K-ras in pancreatic cancer.

The kinase Mirk is overexpressed in many resected pancreatic adenocarcinomas and is amplified in a subset of pancreatic cancer cell lines. Depletion of Mirk has been shown to lead to apoptosis in pancreatic cancer cell lines, and thus to inhibit their clonogenic growth. Mirk is activated by signaling from activated Rac1 to MKK3 in MDCK cells, but the mechanism of activation of Mirk in pancreatic cancers is unknown.

Mirk/Dyrk1b mediates cell survival in rhabdomyosarcomas.

Rhabdomyosarcoma is the most common sarcoma in children and is difficult to treat if the primary tumor is nonresectable or if the disease presents with metastases. The function of the serine/threonine kinase Mirk was investigated in this cancer. Mirk has both growth arrest and survival functions in terminally differentiating skeletal myoblasts.

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/Dyrk1B mediates survival during the differentiation of C2C12 myoblasts.

The kinase Mirk/dyrk1B is essential for the differentiation of C2C12 myoblasts. Mirk reinforces the G0/G1 arrest state in which differentiation occurs by directly phosphorylating and stabilizing p27(Kip1) and destabilizing cyclin D1. We now demonstrate that Mirk is anti-apoptotic in myoblasts.

Mirk/dyrk1B decreases the nuclear accumulation of class II histone deacetylases during skeletal muscle differentiation.

Mirk/dyrk1B is a member of the dyrk/minibrain family of serine/threonine kinases that mediate the transition from growth to differentiation in lower eukaryotes and mammals. Depletion of endogenous Mirk from C2C12 myoblasts by RNA interference blocks skeletal muscle differentiation (Deng, X., Ewton, D.

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.

The cyclin-dependent kinase inhibitor p27Kip1 is stabilized in G(0) by Mirk/dyrk1B kinase.

Elevated levels of the cyclin-dependent kinase (CDK) inhibitor p27 block the cell in G(0)/G(1) until mitogenic signals activate G(1) 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 G(0) in NIH3T3 cells, when it stabilizes p27 by phosphorylating it at Ser-10.

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.

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 p27(kip1) and the G(1)-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 p27(kip1) and cyclin D1.

The stress-activated protein kinases p38 alpha and JNK1 stabilize p21(Cip1) by phosphorylation.

Stress signals activate the SAPK/JNK and p38 MAPK classes of protein kinases, which mediate cellular responses, including steps in apoptosis and the maturation of some cell types. We now show that stress signals initiated by transforming growth factor-beta 1 (TGF-beta 1) induce G(1) arrest through protein stabilization of the CDK inhibitor p21(Cip1). TGF-beta 1 was previously shown to increase p21 protein levels, which in turn mediated G(1) arrest through inactivation of the CDK2-cyclin E complex in HD3 cells (Yan, Z.

Insulin-like growth factor-I has a biphasic effect on colon carcinoma cells through transient inactivation of forkhead1, initially mitogenic, then mediating growth arrest and differentiation.

IGF-I stimulates intestinal cell differentiation after initiating a short proliferative burst, similar to its effect on muscle cell differentiation. Levels of IGF-I attainable in serum (10-20 ng/ml) induced transient growth stimulation of colon carcinoma cells, then growth arrest. When IGF-I functioned as a mitogen, it blocked differentiation.

IGFBP-3 mediates TGF beta 1 proliferative response in colon cancer cells.

Many human tumor cells are resistant to growth inhibition by TGF beta 1. Resistance may be caused by mutations in TGFbeta receptors or in other components of the TGF beta signal transduction systems, or by knockout of the retinoblastoma (Rb) gene, which in fibroblasts converts cellular response to TGF beta 1 from growth inhibition to growth stimulation. Our earlier studies showed such a switch in response to TGF beta 1 occurred in 45% of colon cancers but without deletion of Rb.

Raf-1 activation stimulates proliferation and inhibits IGF-stimulated differentiation in L6A1 myoblasts.

Our previous work has demonstrated that the insulin-like growth factors (IGFs), acting through a single receptor, stimulate both proliferation and differentiation of L6A1 myoblasts. This unique model system has enabled us to closely examine the switch that regulates these two opposing responses. We have previously shown, using specific inhibitors of the IGF-I signal transduction pathway, that the mitogenic response is mediated by the Ras/Raf/MAP kinase pathway and the myogenic response by the PI 3-kinase/p70s6k pathway (Coolican SA, Samuel DS, Ewton DZ, McWade FJ, Florini JR, J Biol Chem 1997; 272: 6653-62).

Modulation of insulin-like growth factor actions in L6A1 myoblasts by insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5: a dual role for IGFBP-5.

We have previously shown that the insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of skeletal muscle cells in culture, and that these actions in L6A1 muscle cells may be modulated by three secreted IGF binding proteins (IGFBPs), IGFBP-4, -5, and -6. Since we found that the temporal expression pattern of IGFBP-4 and IGFBP-5 differed dramatically during the transition from proliferating myoblasts to differentiated myotubes, we undertook the current study to examine the effects of purified IGFBP-4 and IGFBP-5 on IGF-stimulated actions in L6A1 muscle cells. As has been shown for other cell types, we found that IGFBP-4 had only inhibitory actions, inhibiting IGF-I and IGF-II-stimulated proliferation and differentiation.

The mitogenic and myogenic actions of insulin-like growth factors utilize distinct signaling pathways.

It is well established that mitogens inhibit differentiation of skeletal muscle cells, but the insulin-like growth factors (IGFs), acting through a single receptor, stimulate both proliferation and differentiation of myoblasts. Although the IGF-I mitogenic signaling pathway has been extensively studied in other cell types, little is known about the signaling pathway leading to differentiation in skeletal muscle. By using specific inhibitors of the IGF signal transduction pathway, we have begun to define the signaling intermediates mediating the two responses to IGFs.

Growth hormone and the insulin-like growth factor system in myogenesis.

It is very clear that the GH-IGF axis plays a major role in controlling the growth and differentiation of skeletal muscles, as it does virtually all of the tissues in the animal body. One aspect of this control is unquestioned: circulating GH acts on the liver to stimulate expression of the IGF-I and IGFBP3 genes, substantially increasing the levels of these proteins in the circulation. It also seems that GH stimulates expression of IGF-I genes in skeletal muscle, although there are a number of cases in which skeletal muscle IGF-I expression is elevated in the absence of GH.

Stimulation of myogenic differentiation by a neuregulin, glial growth factor 2. Are neuregulins the long-sought muscle trophic factors secreted by nerves?

It has long been known that nerves stimulate growth and maintenance of skeletal muscles in ways not dependent on physical contacts, but numerous attempts to identify and characterize the myotrophic agent(s) secreted by nerves have been unsuccessful. We here suggest that products of the neuregulin gene may be these agents. The neuregulins are a family of proteins made by alternative splicing of a single transcript to give as many as 15 protein products.

Transforming growth factor-beta isoform expression in insulin-like growth factor stimulated myogenesis.

Transforming growth factor betas (TGF-beta s) are the defining members of a super-family of small proteins that are involved in the regulation of development and morphogenesis in a wide array of systems. Previous studies have demonstrated that TGF-beta s both inhibit and, under specialized conditions, induce the differentiation of myoblasts. TGF-beta have been shown to be secreted by mouse C2C12 myoblast cultures undergoing differentiation.

Actions of anabolic hormones and growth factors on cultured neonatal heart cells.

The effects of a series of major anabolic hormones on incorporation of labeled precursors into protein and DNA were measured in cardiac myocytes from neonatal rats. IGF-I, TGF beta, and insulin all stimulated [3H] leucine incorporation into protein; FGF, EGF, triiodothyronine and dexamethasone had little or no effect. The effect of insulin was biphasic, suggesting some stimulation mediated directly by the insulin receptor and some by cross-reaction with the IGF-I receptor.

IGF binding proteins-4, -5 and -6 may play specialized roles during L6 myoblast proliferation and differentiation.

It is well known that IGFs-I and -II stimulate both the proliferation and differentiation of myoblasts, but the role of the IGF binding proteins (IGFBPs) during these processes has not been established. In this study we show that IGF-I analogs with greatly reduced affinity for IGFBPs exhibited about a 10-fold increase in potency in stimulating proliferation (as in other cell types), but up to a 100-fold greater potency than native IGF-I in stimulating L6A1c differentiation. Analysis of conditioned media revealed that L6 cells secrete significant levels of IGFBPs that react with antisera to IGFBP-4, -5 and -6.

IGF-II is more active than IGF-I in stimulating L6A1 myogenesis: greater mitogenic actions of IGF-I delay differentiation.

Mitogens are generally thought to inhibit myogenesis, and many cell biologists have found it hard to interpret observations that the insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of muscle cells in culture. Our previous studies suggested that the Type I IGF receptor mediates these actions. However, IGF-II and insulin treatment caused myoblasts to differentiate much more extensively, suggesting that more complex mechanisms may be involved.