Mirk/dyrk1B Decreases the Nuclear Accumulation of Class II Histone Deacetylases during Skeletal Muscle Differentiation

Abstract

The availability of in vitro culture systems that recapitulate the major features of muscle cell differentiation has allowed much progress toward the elucidation of the molecular basis for muscle maturation and regeneration. An improved understanding of these processes might have significant clinical ramifications, such as the development of strategies to ameliorate damage to skeletal or cardiac muscle. A novel gene cloned in our laboratory (1, 2), the kinase Mirk, has the unusual properties of mediating cell survival in vitro (2), regulating myoblast differentiation following the commitment stage of myogenesis (3), and maintaining untransformed cells in G 0 arrest (4, 5). In C2C12 myoblasts, and in nontransformed epithelial cells and fibroblasts, Mirk elongates the G 0 phase of the cell cycle by stabilizing the CDK 1 inhibitor p27 Kip1 by phosphorylating p27 at serine 10 (4) and maintaining p27 in the nucleus where it acts as a CDK2 inhibitor. Mirk also destabilizes the G 1 cyclin, cyclin D1, by phosphorylation at threonine 288 (5), adjacent to the GSK3␤ site of threonine 286. In addition, Mirk is activated through the stress signaling pathway by the mitogen-activated protein kinase kinase MKK3 (6) and interacts with p38 MAPK (7).Mirk (minibrain-related kinase) is a member of the dyrk/minibrain family of arginine-directed serine/threonine protein kinases (8 -10) and is identical to dyrk1B (11). Mirk is strongly transcriptionally up-regulated under conditions of mitogen deprivation, such as occurs when myoblasts are transferred from growth to differentiation medium. In particular when IGF-1 is eliminated (2, 3) there is an enrichment of active Mirk kinase in G 0 , the cell cycle stage in which the initial steps of differentiation occur. Whereas Mirk protein levels are very low in dividing myoblasts, they are increased at least 10-fold when myoblasts commit to terminal differentiation and are maintained at elevated levels in mature muscle cells (3). This unusual cluster of characteristics suggests the following model. Mirk levels increase under conditions favorable for terminal differentiation when myoblasts arrest in G 0 ; Mirk aids in maintaining G 0 arrest, and elevated amounts of Mirk protein are further activated by cellular stresses to mediate myoblast differentiation.In this report we have investigated whether Mirk mediates myoblast differentiation solely by maintaining G 0 arrest or whether Mirk has an additional role as a transcriptional activator in muscle differentiation. Mirk is also found in normal hepatocytes, and we have shown (6) that Mirk activates the hepatocyte transcription factor HNF1␣ by phosphorylation. In the myoblast system, depletion of Mirk by RNA interference strongly blocked expression of the myogenic regulatory factor myogenin (3), suggesting that Mirk might control myogenin expression. In the current study, we demonstrate that Mirk functions as a transcriptional activator of MEF2C, a known mediator of myogenin expression, by decreasing the nuclear concentration of class II HDACs tha...

Dephosphorylation at a Conserved SP Motif Governs cAMP Sensitivity and Nuclear Localization of Class IIa Histone Deacetylases*

“…Moreover, expression of CaMKIV in HEK293 cells promoted export of HDAC5 but had minimal effects on Ser-279 phosphorylation (data not shown). Mirk/ dyrk1B was shown to phosphorylate Ser-243 of an alternatively spliced isoform corresponding to the N-terminal half of HDAC9 (78). However, expression of this and related kinases did not alter the subcellular localization of class IIa HDACs in our assay conditions (data not shown).…”

“…Additional phosphorylation sites have been identified in class IIa HDACs, including Ser-298 of HDAC4 (36), Ser-253 of the HDAC9 splice variant (78,82), and multiple residues of HDAC4 and HDAC5 reported by phosphoproteomic studies (see the Human Protein Reference Database) (79,80,83,84), suggesting that complex multisite phosphorylation programs may respond to cAMP and other upstream signaling cues (Fig. 8).…”

Class IIa histone deacetylases: regulating the regulators

“…Dyrk1B phosphorylates HDAC5 at a serine residue shared by HDAC4 and HDAC9, but not by HDAC7. Phosphorylation by Dyrk1 reduces their nuclear accumulation and leads to MEF2 activation (Deng et al, 2005). This residue does not correspond to a 14-3-3 binding site and its mutation does not prevent CaMK-induced nuclear export , but it lies within the nuclear localization region of class IIa HDACs.…”

Mirk/Dyrk1B Mediates Survival during the Differentiation of C2C12Myoblasts

“…Depletion of endogenous Mirk by RNA interference blocked the transcription of myogenin and the subsequent myoblast differentiation program (1). Mirk controls the activation of the myogenin transcription factor MEF2 by regulating nuclear accumulation of the MEF2 inhibitors, class II histone deacetylases (2). However, it has been reported that embryonic knockout of Mirk did not block skeletal muscle development in the early embryo (3), suggesting that Mirk may have a more significant function in muscle repair than in initial myogenesis.…”

“…Mirk has also been shown to act as a transcriptional activator (2,12,13) and to inhibit cell motility (14). Because of these actions of Mirk on cell cycle regulators active in G 0 /G 1 during myoblast differentiation, we extended our study of Mirk substrates to p21 Cip1 .…”