Roles of two protein phosphatases, Reg1-Glc7 and Sit4, and glycogen synthesis in regulation of SNF1 protein kinase

The SNF1 protein kinase of Saccharomyces cerevisiae is a member of the SNF1/AMP-activated protein kinase family, which is essential for metabolic control, energy homeostasis, and stress responses in eukaryotes. SNF1 is activated in response to glucose limitation by phosphorylation of Thr210 on the activation loop of the catalytic subunit Snf1. The SNF1 β-subunit contains a glycogen-binding domain that has been implicated in glucose inhibition of Snf1 Thr210 phosphorylation. To assess the role of glycogen, we examined Snf1 phosphorylation in strains with altered glycogen metabolism. A reg1Δ mutant, lacking Reg1-Glc7 protein phosphatase 1, exhibits elevated glycogen accumulation and phosphorylation of Snf1 during growth on high levels of glucose. Unexpectedly, mutations that abolished glycogen synthesis also restored Thr210 dephosphorylation in glucose-grown reg1Δ cells, indicating that elevated glycogen synthesis contributes to activation of SNF1 and that another phosphatase acts on Snf1. We present evidence that Sit4, a type 2A-like protein phosphatase, contributes to dephosphorylation of Snf1 Thr210. Finally, evidence that the effects of glycogen are not mediated by binding to the β-subunit raises the possibility that elevated glycogen synthesis alters glucose metabolism and thereby reduces glucose signaling to the SNF1 pathway.     

Heterotrimer-independent regulation of activation-loop phosphorylation of Snf1 protein kinase involves two protein phosphatases

The SNF1/AMP-activated protein kinases are αβγ-heterotrimers that sense and regulate energy status in eukaryotes. They are activated by phosphorylation of the catalytic Snf1/α subunit, and the Snf4/γ regulatory subunit regulates phosphorylation through adenine nucleotide binding. In Saccharomyces cerevisiae, the Snf1 subunit is phosphorylated on the activation-loop Thr-210 in response to glucose limitation. To assess the requirement of the heterotrimer for regulated Thr-210 phosphorylation, we examined Snf1 and a truncated Snf1 kinase domain (residues 1-309) that has partial Snf1 function. Snf1(1-309) does not interact with the β and Snf4/γ regulatory subunits, and its activity was independent of them in vivo. Phosphorylation of both Snf1 and Snf1(1-309) increased in response to glucose limitation in wild-type cells and in cells lacking β- and Snf4/γ-subunits. These results indicate that glucose regulation of activation-loop phosphorylation can occur by mechanism(s) that function independently of the regulatory subunits. We further show that the Reg1-Glc7 protein phosphatase 1 and Sit4 type 2A-like phosphatase are largely responsible for dephosphorylation of Thr-210 of Snf1(1-309). Together, these findings suggest that these two phosphatases mediate heterotrimer-independent regulation of Thr-210 phosphorylation.     

Angiotensin II-induced cardiomyocyte hypertrophy in vitro is TAK1-dependent and Smad2/3-independent

Cardiac hypertrophy occurs as an adaptation to hypertension but a sustained hypertrophic response can ultimately lead to heart failure. Angiotensin-II (Ang II) is released following hemodynamic overload and stimulates a cardiac hypertrophic response. AngII also increases expression of the regulatory cytokine, transforming growth factor-β1 (TGFβ1), which is also implicated in the cardiac hypertrophic response and can stimulate activation of Smad2/3 as well as TGFβ-activated kinase 1 (TAK1) signaling mediators. To better understand the downstream signaling events in cardiac hypertrophy, we therefore investigated activation of Smad2/3 and TAK1 signaling pathways in response to Ang II and TGFβ1 using primary neonatal rat cardiomyocytes to model cardiac hypertrophic responses. Small interfering RNA (siRNA) knockdown of Smad 2/3 or TAK1 protein or addition of the TGFβ type I receptor inhibitor, SB431542, were used to investigate the role of downstream mediators of TGFβ signaling in the hypertrophic response. Our data revealed that TGFβ1 stimulation leads to cardiomyocyte hypertrophic phenotypes that were indistinguishable from those occurring in response to Ang II. In addition, inhibition of the TGFβ1 type receptor abolished Ang II-induced hypertrophic changes. Furthermore, the hypertrophic response was also prevented following siRNA knockdown of TAK1 protein, but was unaffected by knockdown of Smad2/3 proteins. We conclude that Ang II-induced cardiomyocyte hypertrophy in vitro occurs in a TAK1-dependent, but Smad-independent, manner.