Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles

We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS97xS99). Surprisingly, serine 97 is not appreciably phosphorylated, whereas serine 99 is only a specific substrate for Akt2 but not Akt1 or Akt3. Although wild-type Synip (WT-Synip) undergoes an insulin-stimulated dissociation from Syntaxin4, the Synip serine 99 to phenylalanine mutant (S99F-Synip) is resistant to Akt2 phosphorylation and fails to display insulin-stimulated Syntaxin4 dissociation. Furthermore, overexpression of WT-Synip in 3T3L1 adipocytes had no effect on insulin-stimulated recruitment of glucose transporter 4 (GLUT4) to the plasma membrane, whereas overexpression of S99F-Synip functioned in a dominant-interfering manner by preventing insulin-stimulated GLUT4 recruitment and plasma membrane fusion. These data demonstrate that insulin activation of Akt2 specifically regulates the docking/fusion step of GLUT4-containing vesicles at the plasma membrane through the regulation of Synip phosphorylation and Synip–Syntaxin4 interaction

Renal Threshold for Glucose Re-Absorption in Patients with Latent Autoimmune Diabetes of Adults is Similar to That of Patients with Type 1 Diabetes Mellitus but Lower than That of Patients with Type 2 Diabetes Mellitus

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HER2 G776S mutation promotes oncogenic potential in colorectal cancer cells when accompanied by loss of APC function

Clinical cancer genome sequencing detects oncogenic variants that are potential targets for cancer treatment, but it also detects variants of unknown significance. These variants may interact with each other to influence tumor pathophysiology, however, such interactions have not been fully elucidated. Additionally, the effect of target therapy for those variants also unclarified. In this study, we investigated the biological functions of a HER2 mutation (G776S mutation) of unknown pathological significance, which was detected together with APC mutation by cancer genome sequencing of samples from a colorectal cancer (CRC) patient. Transfection of the HER2 G776S mutation alone slightly increased the kinase activity and phosphorylation of HER2 protein, but did not activate HER2 downstream signaling or alter the cell phenotype. On the other hand, the HER2 G776S mutation was shown to have strong oncogenic potential when loss of APC function was accompanied. We revealed that loss of APC function increased Wnt pathway activity but also increased RAS-GTP, which increased ERK phosphorylation triggered by HER2 G776S transfection. In addition, afatinib, a pan-HER tyrosine kinase inhibitor, suppressed tumor growth in xenografts derived from HER2 G776S-transfected CRC cells. These findings suggest that this HER2 mutation in CRC may be a potential therapeutic target

Disruption of fyn SH3 domain interaction with a proline-rich motif in liver kinase B1 results in activation of AMP-activated protein kinase

Fyn-deficient mice display increased AMP-activated Protein Kinase (AMPK) activity as a result of Fyn-dependent regulation of Liver Kinase B1 (LKB1) in skeletal muscle. Mutation of Fyn-specific tyrosine sites in LKB1 results in LKB1 export into the cytoplasm and increased AMPK activation site phosphorylation. This study characterizes the structural elements responsible for the physical interaction between Fyn and LKB1. Effects of point mutations in the Fyn SH2/SH3 domains and in the LKB1 proline-rich motif on 1) Fyn and LKB1 binding, 2) LKB1 subcellular localization and 3) AMPK phosphorylation were investigated in C2C12 muscle cells. Additionally, novel LKB1 proline-rich motif mimicking cell permeable peptides were generated to disrupt Fyn/LKB1 binding and investigate the consequences on AMPK activity in both C2C12 cells and mouse skeletal muscle. Mutation of either Fyn SH3 domain or the proline-rich motif of LKB1 resulted in the disruption of Fyn/LKB1 binding, re-localization of 70% of LKB1 signal in the cytoplasm and a 2-fold increase in AMPK phosphorylation. In vivo disruption of the Fyn/LKB1 interaction using LKB1 proline-rich motif mimicking cell permeable peptides recapitulated Fyn pharmacological inhibition. We have pinpointed the structural elements within Fyn and LKB1 that are responsible for their binding, demonstrating the functionality of this interaction in regulating AMPK activity

Dapagliflozin rescues endoplasmic reticulum stress-mediated cell death

The new type 2 diabetes drug, dapagliflozin, reduces blood glucose levels and body weight by inhibiting sodium glucose transporter 2 (SGLT2) in proximal tubular cells. SGLT2 inhibitors might modulate glucose influx into renal tubular cells, thereby regulating the metabolic conditions that cause endoplasmic reticulum (ER) stress in the cells. In this study, we examined the effect of dapagliflozin on ER stress in the HK-2 proximal tubular cell line and in the kidney of db/db mice to characterise its function in diabetic nephropathy (DN). We found that dapagliflozin regulated ER stress-mediated apoptosis in vitro and in vivo. Only the elf2α-ATF4-CHOP pathway was regulated under these conditions. Notably, the drug rescued C2 ceramide-induced ER stress-mediated apoptosis and ER stress-mediated apoptosis, which might occur in DN, in db/db mice. Our study shows a novel role for dapagliflozin as an inhibitor of ER stress and suggests that dapagliflozin might be useful for the prevention of DN

Role of Fyn and the interleukin-6-STAT-3-autophagy axis in sarcopenia

Summary: Sarcopenia is the progressive loss of muscle mass wherein Fyn regulates STAT3 to decrease autophagy. To elucidate the role of inflammation in Fyn-STAT3-dependent autophagy and sarcopenia, here we aimed to investigate the underlying mechanisms using two mouse models of primary and secondary sarcopenia: (1) tail suspension and (2) sciatic denervation. In wild-type mice, the expression of Fyn and IL-6 increased significantly. The expression and phosphorylation levels of STAT3 were also significantly augmented, while autophagic activity was abolished. To investigate Fyn-dependency, we used tail suspension with Fyn-null mice. In tail-suspended wild-type mice, IL-6 expression was increased; however, it was abolished in Fyn-null mice, which maintained autophagy and the expression and ablation of STAT3 phosphorylation. In conclusion, Fyn was found to be associated with the IL-6-STAT3-autophagy axis in sarcopenia. This finding permits a better understanding of sarcopenia-associated metabolic diseases and the possible development of therapeutic interventions

LKB1 P328A mutation induces AMPK activation in skeletal muscle.

<p>(A) <i>Tibialis anterior</i> of control mice was transfected with either with pcDNA-Flag-LKB1-WT (right leg) or pcDNA-Flag-LKB1-P328A mutant (left leg). Lysates were prepared and proteins separated by electrophoresis. LKB1, AMPK, phospho- T¹⁷² AMPK, ACC and phospho S⁷⁹-ACC expression levels were determined using specific antibodies. Images represent a single experiment (n = 2 mice) that was repeated 3 times (n = 6 mice). (B) Signal quantification of the expression levels of phospho- T¹⁷² AMPK, and phospho S⁷⁹-ACC from 3 independent experiments</p

<i>In vitro</i> interaction of the SH3 domain of Fyn kinase with the proline-rich domain of LKB1 (A) Purified His-LKB1-WT fusion protein was immobilized onto cobalt beads and incubated either with purified GST protein (control), GST-Fyn-WT or GST-Fyn-W119A fusion protein.

<p>Retained proteins were separated onto SDS-polyacrylamide gel electrophoresis. Binding was detected using anti-GST (detecting Fyn kinase) and anti-His (detecting LKB1) antibodies. Blots are representative of 4 independent experiments. (B) Purified His-LKB1-WT or His-LKB1-P328A mutant fusion protein was immobilized onto cobalt beads and incubated with either purified GST protein (control) or GST-Fyn-WT. Binding was detected using anti-GST (detecting Fyn kinase) and anti-His (detecting LKB1) antibodies. (C) Purified His-LKB1-WT or His-LKB1-P328A mutant were incubated with GST-Fyn-kinase for the indicated time. Phosphorylation of LKB1 was detected by the phospho-tyrosine specific 4G10 antibody. Blots are representative of 3 independent experiments. D. Signal quantification. Identical letters indicate values that are not statistically different from each other (P>0.05).</p