Pathological role of serum- and glucocorticoid-regulated kinase 1 in adverse ventricular remodeling.

BACKGROUND: Heart failure is a growing cause of morbidity and mortality. Cardiac phosphatidylinositol 3-kinase signaling promotes cardiomyocyte survival and function, but it is paradoxically activated in heart failure, suggesting that chronic activation of this pathway may become maladaptive. Here, we investigated the downstream phosphatidylinositol 3-kinase effector, serum- and glucocorticoid-regulated kinase-1 (SGK1), in heart failure and its complications. METHODS AND RESULTS: We found that cardiac SGK1 is activated in human and murine heart failure. We investigated the role of SGK1 in the heart by using cardiac-specific expression of constitutively active or dominant-negative SGK1. Cardiac-specific activation of SGK1 in mice increased mortality, cardiac dysfunction, and ventricular arrhythmias. The proarrhythmic effects of SGK1 were linked to biochemical and functional changes in the cardiac sodium channel and could be reversed by treatment with ranolazine, a blocker of the late sodium current. Conversely, cardiac-specific inhibition of SGK1 protected mice after hemodynamic stress from fibrosis, heart failure, and sodium channel alterations. CONCLUSIONS: SGK1 appears both necessary and sufficient for key features of adverse ventricular remodeling and may provide a novel therapeutic target in cardiac disease.

TTBK2 kinase substrate specificity and the impact of spinocerebellar-ataxia-causing mutations on expression, activity, localization and development.

Mutations that truncate the C-terminal non-catalytic moiety of TTBK2 (tau tubulin kinase 2) cause the inherited, autosomal dominant, SCA11 (spinocerebellar ataxia type 11) movement disorder. In the present study we first assess the substrate specificity of TTBK2 and demonstrate that it has an unusual preference for a phosphotyrosine residue at the +2 position relative to the phosphorylation site. We elaborate a peptide substrate (TTBKtide, RRKDLHDDEEDEAMSIYpA) that can be employed to quantify TTBK2 kinase activity. Through modelling and mutagenesis we identify a putative phosphate-priming groove within the TTBK2 kinase domain. We demonstrate that SCA11 truncating mutations promote TTBK2 protein expression, suppress kinase activity and lead to enhanced nuclear localization. We generate an SCA11-mutation-carrying knockin mouse and show that this leads to inhibition of endogenous TTBK2 protein kinase activity. Finally, we find that, in homozygosity, the SCA11 mutation causes embryonic lethality at embryonic day 10. These findings provide the first insights into some of the intrinsic properties of TTBK2 and reveal how SCA11-causing mutations affect protein expression, catalytic activity, localization and development. We hope that these findings will be helpful for future investigation of the regulation and function of TTBK2 and its role in SCA11.

The structure and regulation of myotubularin phosphatases.

The human neuromuscular diseases X-linked myotubular myopathy and Charcot-Marie-Tooth disease type 4B are caused by mutations in myotubularin family proteins. The myotubularins are a unique subfamily of protein tyrosine phosphatases that utilize inositol phospholipids, rather than phosphoproteins, as substrates. Recent structural studies, including the first crystal structure of a myotubularin family protein, have defined the structural features that are characteristic of the family and revealed the molecular basis of their unique substrate specificity. Interestingly, the myotubularin family contains a subgroup of proteins that are catalytically inactive. Recent biochemical studies have established that the inactive myotubularins function as adaptors for the active members and play an important regulatory role within the family.

Death-associated protein kinase 1 phosphorylates NDRG2 and induces neuronal cell death.

Death-associated protein kinase 1 (DAPK1) has been shown to have important roles in neuronal cell death in several model systems and has been implicated in multiple diseases, including Alzheimer's disease (AD). However, little is known about the molecular mechanisms by which DAPK1 signals neuronal cell death. In this study, N-myc downstream-regulated gene 2 (NDRG2) was identified as a novel substrate of DAPK1 using phospho-peptide library screening. DAPK1 interacted with NDRG2 and directly phosphorylated the Ser350 residue in vitro and in vivo. Moreover, DAPK1 overexpression increased neuronal cell death through NDRG2 phosphorylation after ceramide treatment. In contrast, inhibition of DAPK1 by overexpression of a DAPK1 kinase-deficient mutant and small hairpin RNA, or by treatment with a DAPK1 inhibitor significantly decreased neuronal cell death, and abolished NDRG2 phosphorylation in cell culture and in primary neurons. Furthermore, NDRG2-mediated cell death by DAPK1 was required for a caspase-dependent poly-ADP-ribose polymerase cleavage. In addition, DAPK1 ablation suppressed ceramide-induced cell death in mouse brain and neuronal cell death in Tg2576 APPswe-overexpressing mice. Finally, levels of phosphorylated NDRG2 Ser350 and DAPK1 were significantly increased in human AD brain samples. Thus, phosphorylation of NDRG2 on Ser350 by DAPK1 is a novel mechanism activating NDRG2 function and involved in neuronal cell death regulation in vivo.

A Cross-Species Study of PI3K Protein-Protein Interactions Reveals the Direct Interaction of P85 and SHP2.

Using a series of immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) experiments and reciprocal BLAST, we conducted a fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome in a drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. Using H929 human cancer cells and drosophila S2R+ cells, our data revealed an unexpected direct binding of Corkscrew, the drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines, and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (GAB2). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway.

EGF-receptor specificity for phosphotyrosine-primed substrates provides signal integration with Src.

Aberrant activation of the EGF receptor (EGFR) contributes to many human cancers by activating the Ras-MAPK pathway and other pathways. EGFR signaling is augmented by Src-family kinases, but the mechanism is poorly understood. Here, we show that human EGFR preferentially phosphorylates peptide substrates that are primed by a prior phosphorylation. Using peptides based on the sequence of the adaptor protein Shc1, we show that Src mediates the priming phosphorylation, thus promoting subsequent phosphorylation by EGFR. Importantly, the doubly phosphorylated Shc1 peptide binds more tightly than singly phosphorylated peptide to the Ras activator Grb2; this binding is a key step in activating the Ras-MAPK pathway. Finally, a crystal structure of EGFR in complex with a primed Shc1 peptide reveals the structural basis for EGFR substrate specificity. These results provide a molecular explanation for the integration of Src and EGFR signaling with downstream effectors such as Ras.

Molecular basis for substrate recognition by MTMR2, a myotubularin family phosphoinositide phosphatase.

Myotubularins, a large family of catalytically active and inactive proteins, belong to a unique subgroup of protein tyrosine phosphatases that use inositol phospholipids, rather than phosphoproteins, as physiological substrates. Here, by integrating crystallographic and deuterium-exchange mass spectrometry studies of human myotubularin-related protein-2 (MTMR2) in complex with phosphoinositides, we define the molecular basis for this unique substrate specificity. Phosphoinositide substrates bind in a pocket located on a positively charged face of the protein, suggesting an electrostatic mechanism for membrane targeting. A flexible, hydrophobic helix makes extensive interactions with the diacylglycerol moieties of substrates, explaining the specificity for membrane-bound phosphoinositides. An extensive H-bonding network and charge-charge interactions within the active site pocket determine phosphoinositide headgroup specificity. The conservation of these specificity determinants within the active, but not the inactive, myotubularins provides insight into the functional differences between the active and inactive members.

Crystal structure of a phosphoinositide phosphatase, MTMR2: insights into myotubular myopathy and Charcot-Marie-Tooth syndrome.

Myotubularin-related proteins are a large subfamily of protein tyrosine phosphatases (PTPs) that dephosphorylate D3-phosphorylated inositol lipids. Mutations in members of the myotubularin family cause the human neuromuscular disorders myotubular myopathy and type 4B Charcot-Marie-Tooth syndrome. The crystal structure of a representative member of this family, MTMR2, reveals a phosphatase domain that is structurally unique among PTPs. A series of mutants are described that exhibit altered enzymatic activity and provide insight into the specificity of myotubularin phosphatases toward phosphoinositide substrates. The structure also reveals that the GRAM domain, found in myotubularin family phosphatases and predicted to occur in approximately 180 proteins, is part of a larger motif with a pleckstrin homology (PH) domain fold. Finally, the MTMR2 structure will serve as a model for other members of the myotubularin family and provide a framework for understanding the mechanism whereby mutations in these proteins lead to disease.