Suppression of gluconeogenic gene transcription by SIK1-induced ubiquitination and degradation of CRTC1

Highlights

• SIK1 specifically destabilizes CRTC1 protein.

• S155, S167, S188 and S346 of CRTC1 are critical for destabilization.

• SIK1 suppresses but CRTC1 stimulates gluconeogenic gene expression.

• SIK1 induces RFWD2-mediated and K628-independent ubiquitination of CRTC1.

Abstract

CRTCs are a group of three transcriptional coactivators required for CREB-dependent transcription. CREB and CRTCs are critically involved in the regulation of various biological processes such as cell proliferation, metabolism, learning and memory. However, whether CRTC1 efficiently induces gluconeogenic gene expression and how CRTC1 is regulated by upstream kinase SIK1 remain to be understood. In this work, we demonstrated SIK1-induced phosphorylation, ubiquitination and degradation of CRTC1 in the context of the regulation of gluconeogenesis. CRTC1 protein was destabilized by SIK1 but not SIK2 or SIK3. This effect was likely mediated by phosphorylation at S155, S167, S188 and S346 residues of CRTC1 followed by K48-linked polyubiquitination and proteasomal degradation. Expression of gluconeogenic genes such as that coding for phosphoenolpyruvate carboxykinase was stimulated by CRTC1, but suppressed by SIK1. Depletion of CRTC1 protein also blocked forskolin-induced gluconeogenic gene expression, knockdown or pharmaceutical inhibition of SIK1 had the opposite effect. Finally, SIK1-induced ubiquitination of CRTC1 was mediated by RFWD2 ubiquitin ligase at a site not equivalent to K628 in CRTC2. Taken together, our work reveals a regulatory circuit in which SIK1 suppresses gluconeogenic gene transcription by inducing ubiquitination and degradation of CRTC1. Our findings have implications in the development of new antihyperglycemic agents.

Introduction

cAMP response element (CRE)-binding protein (CREB) is a multifaceted transcription factor regulating the expression of about 4000 target genes [1], which collectively exert a substantial impact on metabolism [2,3], cell proliferation [4], immune response [5], learning and memory [6], as well as other physiological and pathological processes [7]. CREB activity is regulated by two distinct but interconnected mechanisms. First, protein kinase A (PKA)-mediated phosphorylation of CREB at S133 promotes the recruitment of transcriptional coactivators in the family of histone acetyltransferases [8]. Second, CREB activation is achieved through another group of obligate transcriptional coactivators termed CREB-regulated transcriptional coactivator (CRTCs), also known as transducer of regulated CREB activities (TORCs), consisted of three isoforms CRTC1, CRTC2 and CRTC3 [9].

Although the three CRTC isoforms are structurally and functionally related, their tissue distribution patterns are distinct and some of their biological functions are non-redundant. Whereas CRTC2 and CRTC3 are more abundant in the liver, CRTC1 is more concentrated in certain areas of the brain. However, all isoforms are widely expressed but not restricted to particular types of tissue [10]. In addition, their expression can be substantially increased in response to cellular stress and other conditions. For example, CRTC1 expression is highly induced in many cancer cells [11,12] and by hepatitis B virus [13]. CRTC2 is most studied among the three isoforms. Both CRTC2 and CRTC3 are thought to be key regulators of gluconeogenesis [14], glucose uptake [15], energy homeostasis [16,17] and macrophage polarization [5,18]. A role for CRTC2 in lipid metabolism [19,20] and the requirement of CRTC3 for hormonal control of stress response [21] have also been described. In contrast to CRTC2 and CRTC3, CRTC1 has been shown to have a neuronal function. Particularly, CRTC1 is essential for long term memory [22,23], circadian rhythm [24], dendritic growth [25] and neuronal survival after ischemia [26]. CRTC1-null mice are hyperphagic, obese and infertile [28]. They might also develop hepatic steatosis [27]. They can serve as a model for depression [29]. Disruption of CRTC2 in mice results in increased insulin sensitivity [30]. Knockout of mouse CRTC3 leads to resistance to obesity plausibly due to increased energy expenditure and increased β-adrenergic receptor signaling [31]. These phenotypes highlight the unique biological functions of the three CRTC isoforms.

The activity of CRTCs is tightly regulated by phosphorylation. Hyperphosphorylated CRTCs are bound with 14-3-3 proteins in the cytoplasm and their rapid dephosphorylation are required for activation of CREB-dependent transcription [32]. CRTC phosphorylation is catalyzed by salt-inducible kinases (SIKs), which are AMP-activated protein kinase (AMPK)-related kinases regulated by tumor suppressor kinase LKB1 [33]. SIKs comprising three isoforms were initially identified from adrenal glands of rats fed with high-salt diet [34]. They function as master regulators of sodium sensing [35], bone formation [36] and cAMP signaling [37,38]. Particularly, SIK2 directly phosphorylates CRTC2 at S171 [39] and it also phosphorylates p300 to disrupt its acetylation of CRTC2 at K628 [40]. Deacetylated and phosphorylated CRTC2 undergoes ubiquitination at K628 catalyzed by E3 ubiquitin ligase RFWD2, also known as COP1, leading to proteasomal degradation [39]. Although regulation of other CRTC isoforms by SIKs is assumed [14], it remains to be elucidated whether the modification might be isoform-specific. Particularly, whether CRTC1 is phosphorylated and regulated by SIK1 has not been determined experimentally. In this regard, genetic evidence suggests that the regulation of CRTCs by SIKs is not promiscuous [41,42]. Our previous analysis of the role of LKB1 and SIKs in human T-cell leukemia virus type 1 (HTLV-1) transcription suggests that the three SIK isoforms cooperate with each other in the regulation of CRTC activity [43]. SIK1-knockout mice are viable and normoglycemic on regular chow diet, but they exhibit increased sensitivity to insulin and their plasma insulin levels are elevated on high fat diet. They also have high arterial blood pressure [44,45]. Tissue-specific knockout reveals this specific function of SIK1 in skeletal muscle [45]. SIK2-null mice are hyperglycemic and hypertriglyceridemic [46]. Knockout of SIK3 in mice causes dwarfism. The mice are hypoglycemic and hypolipidemic, with increased insulin sensitivity and aberrant circadian rhythms [41,[47], [48], [49]]. Thus, the three SIK isoforms have distinct and related biological functions.

Regulatory phosphorylation of CRTC2 has also been shown to occur at several other sites including S70 [50], S275 [50,51] and S307 [52]. Additional phosphorylation sites on CRTC1 have also been suggested [53]. These findings prompted us to identify additional regulatory sites on CRTC1. Our gain-of-function and loss-of-function experiments in this study revealed SIK1-induced ubiquitination and degradation of CRTC1. The influence of this regulatory mechanism on gluconeogenic gene expression was also assessed.