SIK2 regulates CRTCs, HDAC4 and glucose uptake in adipocytes.

Salt-inducible kinase 2 (SIK2) is an AMP-activated protein kinase (AMPK) related kinase abundantly expressed in adipose tissue. Our aim was to identify molecular targets and functions of SIK2 in adipocytes, and to address the role of PKA-mediated phosphorylation of SIK2 on Ser358. Modulation of SIK2 in adipocytes resulted in altered phosphorylation of CREB-regulated transcription co-activator 2 (CRTC2), CRTC3 and class IIa histone deacetylase 4 (HDAC4). Furthermore, CRTC2, CRTC3, HDAC4 and protein phosphatase 2A (PP2A) interacted with SIK2, and the binding of CRTCs and PP2A to wild-type but not Ser358Ala SIK2, was reduced by cAMP elevation. Silencing of SIK2 resulted in reduced GLUT4 (also known as SLC2A4) protein levels, whereas cells treated with CRTC2 or HDAC4 siRNA displayed increased levels of GLUT4. Overexpression or pharmacological inhibition of SIK2 resulted in increased and decreased glucose uptake, respectively. We also describe a SIK2­CRTC2­HDAC4 pathway and its regulation in human adipocytes, strengthening the physiological relevance of our findings. Collectively, we demonstrate that SIK2 acts directly on CRTC2, CRTC3 and HDAC4, and that the cAMP­PKA pathway reduces the interaction of SIK2 with CRTCs and PP2A. Downstream, SIK2 increases GLUT4 levels and glucose uptake in adipocytes.

LKB1 signalling attenuates early events of adipogenesis and responds to adipogenic cues.

cAMP-response element-binding protein (CREB) is required for the induction of adipogenic transcription factors such as CCAAT/enhancer-binding proteins (C/EBPs). Interestingly, it is known from studies in other tissues that LKB1 and its substrates AMP-activated protein kinase (AMPK) and salt-inducible kinases (SIKs) negatively regulate gene expression by phosphorylating the CREB co-activator CRTC2 and class IIa histone deacetylases (HDACs), which results in their exclusion from the nucleus where they co-activate or inhibit their targets. In this study, we show that AMPK/SIK signalling is acutely attenuated during adipogenic differentiation of 3T3-L1 preadipocytes, which coincides with the dephosphorylation and nuclear translocation of CRTC2 and HDAC4. When subjected to differentiation, 3T3-L1 preadipocytes in which the expression of LKB1 was stably reduced using shRNA (Lkb1-shRNA), as well as Lkb1-knockout mouse embryonic fibroblasts (Lkb1(-/-) MEFs), differentiated more readily into adipocyte-like cells and accumulated more triglycerides compared with scrambled-shRNA-expressing 3T3-L1 cells or Wt MEFs. In addition, the phosphorylation of CRTC2 and HDAC4 was reduced, and the mRNA expression of adipogenic transcription factors Cebpa, peroxisome proliferator-activated receptor γ (Pparg) and adipocyte-specific proteins such as hormone-sensitive lipase (HSL), fatty acid synthase (FAS), aP2, GLUT4 and adiponectin was increased in the absence of LKB1. The mRNA and protein expression of Ddit3/CHOP10, a dominant-negative member of the C/EBP family, was reduced in Lkb1-shRNA-expressing cells, providing a potential mechanism for the up-regulation of Pparg and Cebpa expression. These results support the hypothesis that LKB1 signalling keeps preadipocytes in their non-differentiated form.

Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes.

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes remains elusive. Previous studies have identified LKB1 as a major AMPK kinase in muscle, liver, and other tissues. In certain cell types, Ca(2+) /calmodulin-dependent protein kinase kinase ß (CaMKKß) has been shown to activate AMPK in response to increases of intracellular Ca(2+) levels. Our aim was to investigate if LKB1 and/or CaMKK function as AMPK kinases in adipocytes. We used adipose tissue and isolated adipocytes from mice in which the expression of LKB1 was reduced to 10-20% of that of wild-type (LKB1 hypomorphic mice). We show that adipocytes from LKB1 hypomorphic mice display a 40% decrease in basal AMPK activity and a decrease of AMPK activity in the presence of the AMPK activator phenformin. We also demonstrate that stimulation of 3T3L1 adipocytes with intracellular [Ca(2+) ]-raising agents results in an activation of the AMPK pathway. The inhibition of CaMKK isoforms, particularly CaMKKß, by the inhibitor STO-609 or by siRNAs, blocked Ca(2+) -, but not phenformin-, AICAR-, or forskolin-induced activation of AMPK, indicating that CaMKK activated AMPK in response to Ca(2+) . Collectively, we show that LKB1 is required to maintain normal AMPK-signaling in non-stimulated adipocytes and in the presence of phenformin. In addition, we demonstrate the existence of a Ca(2+) /CaMKK signaling pathway that can also regulate the activity of AMPK in adipocytes.

Inferring signaling pathway topologies from multiple perturbation measurements of specific biochemical species.

The specification of biological decisions by signaling pathways is encoded by the interplay between activation dynamics and network topologies. Although we can describe complex networks, we cannot easily determine which topology the cell actually uses to transduce a specific signal. Experimental testing of all plausible topologies is infeasible because of the combinatorially large number of experiments required to explore the complete hypothesis space. Here, we demonstrate that Bayesian inference-based modeling provides an approach to explore and constrain this hypothesis space,permitting the rational ranking of pathway models. Our approach can use measurements of a limited number of biochemical species when combined with multiple perturbations. As proof of concept, we examined the activation of the extracellular signal-regulated kinase (ERK) pathway by epidermal growth factor. The predicted and experimentally validated model shows that both Raf-1 and, unexpectedly,B-Raf are needed to fully activate ERK in two different cell lines. Thus, our formal methodology rationally infers evidentially supported pathway topologies even when a limited number of biochemical and kinetic measurements are available.

Inferring signaling pathway topologies from multiple perturbation measurements of specific biochemical species.

The specification of biological decisions by signaling pathways is encoded by the interplay between activation dynamics and network topologies. Although we can describe complex networks, we cannot easily determine which topology the cell actually uses to transduce a specific signal. Experimental testing of all plausible topologies is infeasible because of the combinatorially large number of experiments required to explore the complete hypothesis space. Here, we demonstrate that Bayesian inference-based modeling provides an approach to explore and constrain this hypothesis space, permitting the rational ranking of pathway models. Our approach can use measurements of a limited number of biochemical species when combined with multiple perturbations. As proof of concept, we examined the activation of the extracellular signal-regulated kinase (ERK) pathway by epidermal growth factor. The predicted and experimentally validated model shows that both Raf-1 and, unexpectedly, B-Raf are needed to fully activate ERK in two different cell lines. Thus, our formal methodology rationally infers evidentially supported pathway topologies even when a limited number of biochemical and kinetic measurements are available.

Computational modelling of cancerous mutations in the EGFR/ERK signalling pathway.

BACKGROUND: The Epidermal Growth Factor Receptor (EGFR) activated Extracellular-signal Regulated Kinase (ERK) pathway is a critical cell signalling pathway that relays the signal for a cell to proliferate from the plasma membrane to the nucleus. Deregulation of the EGFR/ERK pathway due to alterations affecting the expression or function of a number of pathway components has long been associated with numerous forms of cancer. Under normal conditions, Epidermal Growth Factor (EGF) stimulates a rapid but transient activation of ERK as the signal is rapidly shutdown. Whereas, under cancerous mutation conditions the ERK signal cannot be shutdown and is sustained resulting in the constitutive activation of ERK and continual cell proliferation. In this study, we have used computational modelling techniques to investigate what effects various cancerous alterations have on the signalling flow through the ERK pathway. RESULTS: We have generated a new model of the EGFR activated ERK pathway, which was verified by our own experimental data. We then altered our model to represent various cancerous situations such as Ras, B-Raf and EGFR mutations, as well as EGFR overexpression. Analysis of the models showed that different cancerous situations resulted in different signalling patterns through the ERK pathway, especially when compared to the normal EGF signal pattern. Our model predicts that cancerous EGFR mutation and overexpression signals almost exclusively via the Rap1 pathway, predicting that this pathway is the best target for drugs. Furthermore, our model also highlights the importance of receptor degradation in normal and cancerous EGFR signalling, and suggests that receptor degradation is a key difference between the signalling from the EGF and Nerve Growth Factor (NGF) receptors. CONCLUSION: Our results suggest that different routes to ERK activation are being utilised in different cancerous situations which therefore has interesting implications for drug selection strategies. We also conducted a comparison of the critical differences between signalling from different growth factor receptors (namely EGFR, mutated EGFR, NGF, and Insulin) with our results suggesting the difference between the systems are large scale and can be attributed to the presence/absence of entire pathways rather than subtle difference in individual rate constants between the systems.

Protein kinase B activity is required for the effects of insulin on lipid metabolism in adipocytes.

Protein kinase B (PKB) is known to mediate a number of biological responses to insulin and growth factors, its role in glucose uptake being one of the most extensively studied. In this work, we have employed a recently described allosteric inhibitor of PKB, Akti, to clarify the role of PKB in lipid metabolism in adipocytes-a subject that has received less attention. Pretreatment of primary rat and 3T3L1 adipocytes with Akti resulted in dose-dependent inhibition of PKB phosphorylation and activation in response to insulin, without affecting upstream insulin signaling [insulin receptor (IR), insulin receptor substrate (IRS)] or the insulin-induced phosphoinositide 3-kinase (PI3K)-dependent activation of the ERK/p90 ribosomal kinase (RSK) pathway. PKB activity was required for the insulin-induced activation of phosphodiesterase 3B (PDE3B) and for the antilipolytic action of insulin. Moreover, inhibition of PKB activity resulted in a reduction in de novo lipid synthesis and in the ability of insulin to stimulate this process. The regulation of the rate-limiting lipogenic enzyme acetyl-CoA carboxylase (ACC) by insulin through dephosphorylation of S79, which is a target for AMP-activated protein kinase (AMPK), was dependent on the presence of active PKB. Finally, AMPK was shown to be phosphorylated by PKB on S485 in response to insulin, and this was associated with a reduction in AMPK activity. In summary, we propose that PKB is required for the positive effects of insulin on lipid storage and that regulation of PDE3B and AMPK by PKB is important for these effects.