Literature context: Cell Signaling Technology 9624; RRID:AB_331817 Anti-Flag Sigma F3165; RRID: AB
Nutrient deprivation induces autophagy through inhibiting TORC1 activity. We describe a novel mechanism in Drosophila by which TORC1 regulates RNA processing of Atg transcripts and alters ATG protein levels and activities via the cleavage and polyadenylation (CPA) complex. We show that TORC1 signaling inhibits CDK8 and DOA kinases, which directly phosphorylate CPSF6, a component of the CPA complex. These phosphorylation events regulate CPSF6 localization, RNA binding, and starvation-induced alternative RNA processing of transcripts involved in autophagy, nutrient, and energy metabolism, thereby controlling autophagosome formation and metabolism. Similarly, we find that mammalian CDK8 and CLK2, a DOA ortholog, phosphorylate CPSF6 to regulate autophagy and metabolic changes upon starvation, revealing an evolutionarily conserved mechanism linking TORC1 signaling with RNA processing, autophagy, and metabolism.
Literature context: Signaling Technology Cat# 9624; RRID:AB_331817 Donkey anti-rabbit IgG, HRP-lin
The lineage-specific transcription factor (TF) MEF2C is often deregulated in leukemia. However, strategies to target this TF have yet to be identified. Here, we used a domain-focused CRISPR screen to reveal an essential role for LKB1 and its Salt-Inducible Kinase effectors (SIK3, in a partially redundant manner with SIK2) to maintain MEF2C function in acute myeloid leukemia (AML). A key phosphorylation substrate of SIK3 in this context is HDAC4, a repressive cofactor of MEF2C. Consequently, targeting of LKB1 or SIK3 diminishes histone acetylation at MEF2C-bound enhancers and deprives leukemia cells of the output of this essential TF. We also found that MEF2C-dependent leukemias are sensitive to on-target chemical inhibition of SIK activity. This study reveals a chemical strategy to block MEF2C function in AML, highlighting how an oncogenic TF can be disabled by targeting of upstream kinases.
Literature context: ll Signaling Technologies 9624; RRID:AB_331817 Rabbit anti-GAPDH Cell Signalin
Although the cAMP-dependent protein kinase (PKA) is ubiquitously expressed, it is sequestered at specific subcellular locations throughout the cell, thereby resulting in compartmentalized cellular signaling that triggers site-specific behavioral phenotypes. We developed a three-step engineering strategy to construct an optogenetic PKA (optoPKA) and demonstrated that, upon illumination, optoPKA migrates to specified intracellular sites. Furthermore, we designed intracellular spatially segregated reporters of PKA activity and confirmed that optoPKA phosphorylates these reporters in a light-dependent fashion. Finally, proteomics experiments reveal that light activation of optoPKA results in the phosphorylation of known endogenous PKA substrates as well as potential novel substrates.
Literature context: at# 9624; RRID:AB_331817 Anti-pCaMK
Elucidating temporal windows of signaling activity required for synaptic and behavioral plasticity is crucial for understanding molecular mechanisms underlying these phenomena. Here, we developed photoactivatable autocamtide inhibitory peptide 2 (paAIP2), a genetically encoded, light-inducible inhibitor of CaMKII activity. The photoactivation of paAIP2 in neurons for 1-2 min during the induction of LTP and structural LTP (sLTP) of dendritic spines inhibited these forms of plasticity in hippocampal slices of rodents. However, photoactivation ∼1 min after the induction did not affect them, suggesting that the initial 1 min of CaMKII activation is sufficient for inducing LTP and sLTP. Furthermore, the photoactivation of paAIP2 expressed in amygdalar neurons of mice during an inhibitory avoidance task revealed that CaMKII activity during, but not after, training is required for the memory formation. Thus, we demonstrated that paAIP2 is useful to elucidate the temporal window of CaMKII activation required for synaptic plasticity and learning.
Literature context: lÂ Â 1:1000Â Phospho-PKA substrateÂ AB_331817Â CST, 9624SÂ Rabbit; monoclonalÂ Â
Obesity has become the most common metabolic disorder worldwide. Promoting brown adipose tissue (BAT) and beige adipose tissue formation, and therefore, a functional increase in energy expenditure, may counteract obesity. Mice lacking type IIβ regulatory subunit of adenosine 3',5' cyclic monophosphate (cAMP)-dependent protein kinase A (PKA-RIIB) display reduced adiposity and resistance to diet-induced obesity. PKA-RIIB, encoded by the Prkar2b gene, is most abundant in BAT and white adipose tissue (WAT) and in the brain. In this study, we show that mice lacking PKA-RIIB have increased energy expenditure, limited weight gain, and improved glucose metabolism. PKA-RIIB deficiency induces brownlike adipocyte in inguinal WAT (iWAT). PKA-RIIB deficiency also increases the expression of uncoupling protein 1 and other thermogenic genes in iWAT and primary preadipocytes from iWAT through a mechanism involving increased PKA activity, which is represented by increased phosphorylation of PKA substrate, cAMP response element binding protein, and P38 mitogen-activated protein kinase. Our study provides evidence for the role of PKA-RIIB deficiency in regulating thermogenesis in WAT, which may potentially have therapeutic implications for the treatment of obesity and related metabolic disorders.
Literature context: # 9624s; RRID:AB_331817 Rabbit mon
Glucagon and thyroid hormone (T3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease.
Protein kinase A (PKA) regulates osteoblast cell function in vitro and is activated by important bone mass modulating agents. We determined whether PKA activation in osteoblasts is sufficient to mediate a bone anabolic response. Thus, a mouse model conditionally expressing a constitutively active PKA (CA-PKA) in osteoblasts (CA-PKA-OB mouse) was developed by crossing a 2.3-kb α1 (I)-collagen promoter-Cre mouse with a floxed-CA-PKA mouse. Primary osteoblasts from the CA-PKA-OB mice exhibited higher basal PKA activity than those from control mice. Microcomputed tomographic analysis revealed that CA-PKA-OB female mice had an 8.6-fold increase in femoral but only 1.16-fold increase in lumbar 5 vertebral bone volume/total volume. Femur cortical thickness and volume were also higher in the CA-PKA-OB mice. In contrast, alterations in many femoral microcomputed tomographic parameters in male CA-PKA-OB mice were modest. Interestingly, the 3-dimensional structure model index was substantially lower both in femur and lumbar 5 of male and female CA-PKA-OB mice, reflecting an increase in the plate to rod-like structure ratio. In agreement, femurs from female CA-PKA-OB mice had greater load to failure and were stiffer compared with those of control mice. Furthermore, the CA-PKA-OB mice had higher levels of serum bone turnover markers and increased osteoblast and osteoclast numbers per total tissue area compared with control animals. In summary, constitutive activation of PKA in osteoblasts is sufficient to increase bone mass and favorably modify bone architecture and improve mechanical properties. PKA activation in mature osteoblasts is, therefore, an important target for designing anabolic drugs for treating diseases with bone loss.