Dual phosphorylation of Sin1 at T86 and T398 negatively regulates mTORC2 complex integrity and activity

Mammalian target of rapamycin (mTOR) plays essential roles in cell proliferation, survival and metabolism by forming at least two functional distinct multi-protein complexes, mTORC1 and mTORC2. External growth signals can be received and interpreted by mTORC2 and further transduced to mTORC1. On the other hand, mTORC1 can sense inner-cellular physiological cues such as amino acids and energy states and can indirectly suppress mTORC2 activity in part through phosphorylation of its upstream adaptors, IRS-1 or Grb10, under insulin or IGF-1 stimulation conditions. To date, upstream signaling pathways governing mTORC1 activation have been studied extensively, while the mechanisms modulating mTORC2 activity remain largely elusive. We recently reported that Sin1, an essential mTORC2 subunit, was phosphorylated by either Akt or S6K in a cellular context-dependent manner. More importantly, phosphorylation of Sin1 at T86 and T398 led to a dissociation of Sin1 from the functional mTORC2 holo-enzyme, resulting in reduced Akt activity and sensitizing cells to various apoptotic challenges. Notably, an ovarian cancer patient-derived Sin1-R81T mutation abolished Sin1-T86 phosphorylation by disrupting the canonical S6K-phoshorylation motif, thereby bypassing Sin1-phosphorylation-mediated suppression of mTORC2 and leading to sustained Akt signaling to promote tumorigenesis. Our work therefore provided physiological and pathological evidence to reveal the biological significance of Sin1 phosphorylation-mediated suppression of the mTOR/Akt oncogenic signaling, and further suggested that misregulation of this process might contribute to Akt hyper-activation that is frequently observed in human cancers.

LRRK2 phosphorylates Snapin and inhibits interaction of Snapin with SNAP-25

Leucine-rich repeat kinase 2 (LRRK2) is a gene that, upon mutation, causes autosomal-dominant familial Parkinson's disease (PD). Yeast two-hybrid screening revealed that Snapin, a SNAP-25 (synaptosomal-associated protein-25) interacting protein, interacts with LRRK2. An in vitro kinase assay exhibited that Snapin is phosphorylated by LRRK2. A glutathione-S-transferase (GST) pull-down assay showed that LRRK2 may interact with Snapin via its Ras-of-complex (ROC) and N-terminal domains, with no significant difference on interaction of Snapin with LRRK2 wild type (WT) or its pathogenic mutants. Further analysis by mutation study revealed that Threonine 117 of Snapin is one of the sites phosphorylated by LRRK2. Furthermore, a Snapin T117D phosphomimetic mutant decreased its interaction with SNAP-25 in the GST pull-down assay. SNAP-25 is a component of the SNARE (Soluble NSF Attachment protein REceptor) complex and is critical for the exocytosis of synaptic vesicles. Incubation of rat brain lysate with recombinant Snapin T117D, but not WT, protein caused decreased interaction of synaptotagmin with the SNARE complex based on a co-immunoprecipitation assay. We further found that LRRK2-dependent phosphorylation of Snapin in the hippocampal neurons resulted in a decrease in the number of readily releasable vesicles and the extent of exocytotic release. Combined, these data suggest that LRRK2 may regulate neurotransmitter release via control of Snapin function by inhibitory phosphorylation.

Searching for the role of protein phosphatases in eukaryotic microorganisms

Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs) into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively). Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism

Clonagem e caracterização da serina/treonina fosfatase tipo 1(PP1) de Dictyostelium discoideum / Cloning and characterization of the serine/threonine phosphatase type 1(PP1) of Dictyostelium discoideum

A serina/treonina fosfatase tipo 1 (PP1) é uma enzima importante para diversos processos celulares nos eucariotos. Apesar disso, a atividade enzimática da PP1 nunca foi detectada em extratos celulares de Dictyostelium discoideum, ao contrário da atividade da enzima PP2A, que é outra proteína fosfatase de ocorrência geral nos eucariotos. Nesse estudo repostamos a clonagem e o sequenciamento de um cDNA codificador para a subunidade catalílitica da PP1 de D. discoideum. Verificamos que esse cDNA tem origem a partir de um gene em cópia única, que expressa uma proteína ao longo de todo o ciclo de vida desse organismo. A dedução da seqüência da proteína a partir do cDNA revela uma identidade de 80 por cento em média com as seqüências de outras PP1, sendo a principal diferença observada a substituição da cisteína 269 por uma fenilalanina...

The Effect of Disodium Etidronate on Phosphatase Activity in Experimental bone Formation of Rats / 대한정형외과학회잡지

The bone formation of periarticular connective tissue after head injury and total hip arthroplasty is included in the category of heterotopic ossification. Induction of a new bone formation in the soft tissue is related to various materials such as bone morphogenic protein. The alkaline phosphatase and acid phosphatase act as important factors in the formation and absorption of the bone. The acid phospatase has the important function of acting as the control with specific activity of phosphatase in vivo. Cholecalciferol induces absorption of the calcium in the alimentary tract and bone resorption and increment of bone calcification, whereas disodium etidronate inhibits the deposition and dissolution of calcium salt and formation of heterotopic bone. This paper reports on the relationship of alkaline phosphatase and various phosphoaminoacid phosphatase which affect the cellular differentiation and remodelling in the heterotopic ossification, with the effect of cholecalciferol and disodium etidronate on the heterotopic bone induction in rats. The following results were obtained: 1. The contents of the calcium in the implanted bone matrix increased markedly from two to five weeks. There was no changes in the calcium content by cholecalciferol or in the administration of small doses of disodium etidronate (5mg/kg). However, in the administration of large dose of disodium etidronate (25mg/kg), calcium mobilization was totally suppressed for the whole period of the experiment. 2. The protein content in the implanted bone matrix did not much change for the whole period of the experiment and the administratinn of cholecalciferol or disodium etidronate also had no effect on the protein content. 3. The activities of alkaline phosphatase in the implanted bone matrix peaked at two weeks in control or cholecalciferol group, whereas disodium etidronate admninstration caused the highest activity in the third week. 4. The activity of acid phosphatase in the implanted bone matrix increased in first and third weeks by cholecalciferol treatment. Disoidum etidronate inhibited the activity of the acid phosphatase in the first, fourth & sixth weeks of implantation. 5. The activity of phosphoserine phosphatase increased due to cholecalciferol treatment, but was significantly inhibited by disodium etidronate (25mg/kg) treatment. 6. The activity of phosphothreonine phosphatase in the implanted bone matrix slightly increased due to cholecalciferol treatment, whereas the activity decreased significantly for the whole period of the experiment by disodium etidronate (25mg/kg) treatment. 7. The activity of phosphotyrosine phosphatase in the implanted bone matrix was not change much for the whole period of the experiment and the administration of cholecalciferol or disodium etidronate had no effect on the activity of phosphotyrosine phosphatase. In conclusion, the disodium etidronate (25mg/kg) almost completely inhibited the molilization of calcium and the activities of acid phosphatase, phosphoserine and phosphothreonine phosphatases. Therefore, it can be suggested that the above phosphatases are closely related to the action mechanism of disodium etidronate.