Regulation of SIK1 abundance and stability is critical for myogenesis

Abstract

cAMP signaling can both promote and inhibit myogenic differentiation, but little is known about the mechanisms mediating promyogenic effects of cAMP. We previously demonstrated that the cAMP response element-binding protein (CREB) transcriptional target salt-inducible kinase 1 (SIK1) promotes MEF2 activity in myocytes via phosphorylation of class II histone deacetylase proteins (HDACs). However, it was unknown whether SIK1 couples cAMP signaling to the HDAC-MEF2 pathway during myogenesis and how this response could specifically occur in differentiating muscle cells. To address these questions, we explored SIK1 regulation and function in muscle precursor cells before and during myogenic differentiation. We found that in primary myogenic progenitor cells exposed to cAMP-inducing agents, Sik1 transcription is induced, but the protein is rapidly degraded by the proteasome. By contrast, sustained cAMP signaling extends the half-life of SIK1 in part by phosphorylation of Thr475, a previously uncharacterized site that we show can be phosphorylated by PKA in cell-free assays. We also identified a functional PEST domain near Thr475 that contributes to SIK1 degradation. During differentiation of primary myogenic progenitor cells, when PKA activity has been shown to increase, we observe elevated Sik1 transcripts as well as marked accumulation and stabilization of SIK1 protein. Depletion of Sik1 in primary muscle precursor cells profoundly impairs MEF2 protein accumulation and myogenic differentiation. Our findings support an emerging model in which SIK1 integrates cAMP signaling with the myogenic program to support appropriate timing of differentiation. M yoblast differentiation is driven by a transcriptional cascade that is subject to precise temporal control during development as well as after acute muscle injury (1). The second messenger cAMP and its primary effector PKA are known to contribute to myoblast proliferation in the developing dermomyotome (2), to promote migration and fusion of muscle precursor cells (3, 4), and to exert potent anabolic effects on adult skeletal muscle (5). During differentiation of myoblasts ex vivo, intracellular cAMP peaks transiently before cell-cell fusion (6, 7), and PKA signaling is required for expression of differentiation markers in cultured myoblasts (8) and in mouse embryos (2). However, sustained cAMP-PKA signaling prevents myogenic differentiation, in part through inhibition of basic helix-loop-helix and MADS transcription factors (9-11). The mechanisms by which a transient peak of cAMP promotes myoblast differentiation are still poorly understood. Because ligands that induce cAMP production promote muscle regeneration (5), it is important to identify promyogenic cAMP signaling effectors.The transcription factor CREB (cAMP response elementbinding protein) is a key effector of cAMP-PKA in many cell types (12). CREB phosphorylation is induced during early stages of vertebrate myogenesis (2, 13), and CREB activity is required in mice for dermomyotome development (2). We previously...

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“…Next, we purified a GST-tagged kinase-dead Wnd (GST-Wnd KD —K188A, to rule out autophosphorylation of Wnd) from E. coli and evaluated its phosphorylation by MTORC1 in vitro (Stewart et al, 2013). We found that GST-Wnd KD was phosphorylated only in the presence of MTORC1, and Torin1 significantly decreased this phosphorylation (Figures 5H–5I).…”

14‐3‐3 proteins mediate inhibitory effects of cAMP on salt‐inducible kinases (SIKs)

“…Consistent with the inhibitory effect of cAMP/PKA on SIK activity, short-term exposure to the adenylyl cyclase activator Forskolin (Fsk) triggered the dephosphorylation of CRTC2/3, in control and SIK1-3 overexpressing HEK293T cells. In keeping with its short half-life, protein amounts of overexpressed SIK1 were decreased in comparison to SIK2 and SIK3 [34].…”

Lkb1 Is Indispensable for Skeletal Muscle Development, Regeneration, and Satellite Cell Homeostasis

“…Moreover, the myopathy was not rescued by rapamycin‐mediated inhibition of mTOR (data not shown), indicating that activation of the mTOR pathway is not responsible for the muscle pathology in the MyoD‐Lkb1 mice, and other downstream targets of Lkb1 may have mediated the phenotype. For example, the Lkb1 substrate SIK1 has been shown to regulate myogenesis through the class II HDACs . Further studies are needed to elucidate the exact mechanism that lead to the myopathy in the MyoD‐Lkb1 mice.…”