Association of Phosphorylated Pyruvate Dehydrogenase with Pyruvate Kinase M2 Promotes PKM2 Stability in Response to Insulin.

Insulin is a crucial signalling molecule that primarily functions to reduce blood glucose levels through cellular uptake of glucose. In addition to its role in glucose homeostasis, insulin has been shown to regulate cell proliferation. Specifically, insulin enhances the phosphorylation of pyruvate dehydrogenase E1α (PDHA1) at the Ser293 residue and promotes the proliferation of HepG2 hepatocellular carcinoma cells. Furthermore, we previously observed that p-Ser293 PDHA1 bound with pyruvate kinase M2 (PKM2) as confirmed by coimmunoprecipitation. In this study, we used an in silico analysis to predict the structural conformation of the two binding proteins. However, the function of the protein complex remained unclear. To investigate further, we treated cells with si-PDHA1 and si-PKM2, which led to a reduction in PKM2 and p-Ser293 PDHA1 levels, respectively. Additionally, we found that the PDHA S293A dephospho-mimic reduced PKM2 levels and its associated enzyme activity. Treatment with MG132 and leupeptin impeded the PDHA1 S293A-mediated PKM2 reduction. These results suggest that the association between p-PDHA1 and PKM2 promotes their stability and protects them from protein degradation. Of interest, we observed that p-PDHA1 and PKM2 were localized in the nucleus in liver cancer patients. Under insulin stimulation, the knockdown of both PDHA1 and PKM2 led to a reduction in the expression of common genes, including KDMB1. These findings suggest that p-PDHA1 and PKM2 play a regulatory role in these proteins' expression and induce tumorigenesis in response to insulin.

Cross-Talk between Wnt Signaling and Src Tyrosine Kinase.

Src, a non-receptor tyrosine kinase, was first discovered as a prototype oncogene and has been shown to critical for cancer progression for a variety of tissues. Src activity is regulated by a number of post-translational modifications in response to various stimuli. Phosphorylations of Src Tyr419 (human; 416 in chicken) and Src Tyr530 (human; 527 in chicken) have been known to be critical for activation and inactivation of Src, respectively. Wnt signaling regulates a variety of cellular functions including for development and cell proliferation, and has a role in certain diseases such as cancer. Wnt signaling is carried out through two pathways: ß-catenin-dependent canonical and ß-catenin-independent non-canonical pathways as Wnt ligands bind to their receptors, Frizzled, LRP5/6, and ROR1/2. In addition, many signaling components including Axin, APC, Damm, Dishevelled, JNK kinase and Rho GTPases contribute to these canonical and non-canonical Wnt pathways. However, the communication between Wnt signaling and Src tyrosine kinase has not been well reviewed as Src regulates Wnt signaling through LRP6 tyrosine phosphorylation. GSK-3ß phosphorylated by Wnt also regulates Src activity. As Wnt signaling and Src mutually regulate each other, it is noted that aberrant regulation of these components give rise to various diseases including typically cancer, and as such, merit a closer look.

RhoA GTPase phosphorylated at tyrosine 42 by src kinase binds to ß-catenin and contributes transcriptional regulation of vimentin upon Wnt3A.

In the Wnt canonical pathway, Wnt3A has been known to stabilize ß-catenin. In the non-canonical Wnt signaling pathway, Wnt is known to activate Rho GTPases. The correlation between canonical and non-canonical pathways by Wnt signaling, however, has not been well elucidated. Here, we identified that Wnt3A promoted superoxide generation, leading to Tyr42 phosphorylation of RhoA through activations of c-Src and Rho-dependent coiled coil kinase 2 (ROCK2) and phosphorylation of p47phox, a component of NADPH oxidase. Wnt3A also induced accumulation of ß-catenin along with activations of RhoA and ROCK1. Concurrently, ROCK1 was able to phosphorylate GSK-3ß at Ser9, which phosphorylated Src at Ser51 and Ser492 residues, leading to Src inactivation through dephosphorylation of Tyr416 during the late period of Wnt3A treatment. Meanwhile, p-Tyr42 RhoA bound to ß-catenin via the N-terminal domain of ß-catenin, thereby leading to the nuclear translocation of p-Tyr42 RhoA/ß-catenin complex. Notably, p-Tyr42 RhoA as well as ß-catenin was associated with the promoter of Vim, leading to increased expression of vimentin. In addition, stomach cancer patients harboring higher expressed p-Tyr42 Rho levels revealed the much poorer survival probability. Therefore, we propose that p-Tyr42 RhoA is crucial for transcriptional regulation of specific target genes in the nucleus by binding to their promoters and involved in tumorigenesis.

Tyr42 phosphorylation of RhoA GTPase promotes tumorigenesis through nuclear factor (NF)-κB.

Dysregulation of reactive oxygen species (ROS) levels is implicated in the pathogenesis of several diseases, including cancer. However, the molecular mechanisms for ROS in tumorigenesis have not been well established. In this study, hydrogen peroxide activated nuclear factor-κB (NF-κB) and RhoA GTPase. In particular, we found that hydrogen peroxide lead to phosphorylation of RhoA at Tyr42 via tyrosine kinase Src. Phospho-Tyr42 (p-Tyr42) residue of RhoA is a binding site for Vav2, a guanine nucleotide exchange factor (GEF), which then activates p-Tyr42 form of RhoA. P-Tyr42 RhoA then binds to IκB kinase γ (IKKγ), leading to IKKß activation. Furthermore, RhoA WT and phospho-mimic RhoA, RhoA Y42E, both promoted tumorigenesis, whereas the dephospho-mimic RhoA, RhoA Y42F suppressed it. In addition, hydrogen peroxide induced NF-κB activation and cell proliferation, along with expression of c-Myc and cyclin D1 in the presence of RhoA WT and RhoA Y42E, but not RhoA Y42F. Indeed, levels of p-Tyr42 Rho, p-Src, and p-65 are significantly increased in human breast cancer tissues and show correlations between each of the two components. Conclusively, the posttranslational modification of as RhoA p-Tyr42 may be essential for promoting tumorigenesis in response to generation of ROS.