Cell Regulation by small GTPases and Protein Kinases

This research program seeks to identify the molecular structure, function and regulation of elements that mediate signal transduction initiated by the insulin receptor, related receptor tyrosine kinases and counter-acting, anti-insulin signaling pathways. As elucidation of the signaling pathways responsive to insulin progressed, it became evident that many of the effectors identified also participated in the regulation of mitogenic and cell differentiation programs:

Elucidation of the TOR pathway. TOR is a giant protein kinase that responds to nutrient sufficiency and insulin/IGF-1 (and other mitogens) to control the growth/size of all cells (e.g., pancreatic beta cells), and the proliferation of some, such as T cells and vascular smooth muscle cells. TOR also negatively regulates insulin signaling to PI-3 kinase. TOR functions in two physically separate, independently regulated hetero-oligomeric complexes (mTOR complexes 1 and 2); The major effort is directed at understanding how the small, ras-like GTPase, Rheb, which we showed acts directly on the mTOR polypeptide, controls the kinase activity of mTORC1. The activity of mTORC1 is also controlled by amino acids especially leucine. We showed that leucine controls the interaction of Rheb with mTOR in vivo; the basis for this control appears to be mediated by another set of small GTPases, the Rag polypeptides, and current effort is focused on the regulation of the latter. mTOR interaction with its substrates (e.g., 4E-BP, S6K1) appears to be regulated; the basis for this regulation is under study. Finally, the mechanisms by which mTOR gates the translation of the IGF2 mRNA isoform specifically associated with IMP2, as well as the identification of the cohort of mRNAs bound to IMP2 is under investigation; IMP2 is a candidate T2DM locus and fetal IGF2 is relevant to the nutrient control of fetal growth.

The regulation and physiologic functions of the Mst1 and Mst2 protein kinases, which are the mammalian orthologs of the “hippo” kinase, a central element in an anti-proliferative developmental pathway defined in Drosophila. We identified Mst1/2 as constitutive partners of the tumor suppressor proteins RASSF1/Nore1; the latter bind specifically to the active forms of the Ras and Rap1 small GTPases. Transfection and biochemical analyses defined the regulation of the Nore1/Mst1 complex by ras-like GTPases and identified the cyclin-like polypeptide Mob1 as a preferred Mt1/2 substrate. Nevertheless, studies in cell culture proved uninformative as to physiologic functions, impelling the generation of Mst1 and Mst2 KO mice. The Mst1 KO mice exhibit major abnormalities in the function of T cells; elimination of Mst1 greatly enhances the proliferative response of mature, naïve T cells to stimulation of the T cell antigen receptor, whereas the clustering and activation of integrins is severely impaired. These actions of Mst1 are independent of the downstream elements of the canonical “hippo” pathway. The combined deletion of Mst1 and Mst2 from the hematopoietic compartment results in the absence of all peripheral lymphocytes and a profound immunodeficiency syndrome. The elimination of Mst2 results in a major brain phenotype currently under study, and the Mst1/Mst2 double knockout is an early embryonic lethal. Mice that lack both alleles of Mst1 and are heterozygous for Mst2 spontaneously develop hepatocellular carcinoma (HCC) with high penetrance after ~ 6months age; Adeno-Cre-mediated elimination of the conditional Mst2 allele from the liver of Mst1 null mice causes the liver to double in size within one week, and is invariably followed by the development of HCC within eight weeks, effects that reflect in part a loss of negative regulation of the Yap1 coactivator. The liver phenotype reflects an essential, overlapping function of Mst1 and Mst2 in the negative regulation of hepatocyte proliferation, whereas the T cell phenotype reflects predominantly the loss of Mst1/2-mediated regulation of cell adhesion and migration. The identification of the molecular elements upstream and downstream of the Mst1/2 kinases for each of these functions is the focus of present effort.

References:

1. Avruch J, Long X, Lin Y, Ortiz-Vega S, Rapley J, Papageorgiou A, Oshiro N, Kikkawa U. Activation of mTORC1 in two steps: Rheb-GTP activation of catalytic function and increased binding of substrates to raptor. Biochem Soc Trans. 2009;37:223-6.
   
   
2. Avruch J, Xavier R, Bardeesy N, Zhang XF, Praskova M, Zhou D, Xia F. Rassf family of tumor suppressor polypeptides. J Biol Chem. 2009;284:11001-5.
   
   
3. Zhou D, Medoff BD, Chen L, Li L, Zhang XF, Praskova M, Liu M, Landry A, Blumberg RS, Boussiotis VA, Xavier R, Avruch J.The Nore1B/Mst1 complex restrains antigen receptor-induced proliferation of naïve T cells. Proc Natl Acad Sci U S A. 2008;105:20321-6.
   
   
4. Patursky-Polischuk I, Stolovich-Rain M, Hausner-Hanochi M, Kasir J, Cybulski N, Avruch J, Rüegg MA, Hall MN, Meyuhas O. The TSC-mTOR pathway mediates translational activation of TOP mRNAs by insulin largely in a raptor- or rictor-independent manner. Mol Cell Biol. 2009;29:640-9.
   
   
5. Rapley J, Nicolàs M, Groen A, Regué L, Bertran MT, Caelles C, Avruch J, Roig J. The NIMA-family kinase Nek6 phosphorylates the kinesin Eg5 at a novel site necessary for mitotic spindle formation. J Cell Sci. 2008 ;121:3912-21.
   
   
6. Anguera MC, Liu M, Avruch J, Lee JT. Characterization of two Mst1-deficient mouse models. Dev Dyn. 2008 ;237:3424-34.
   
   
7. Avruch J, Long X, Ortiz-Vega S, Rapley J, Papageorgiou A, Dai N. Amino acid regulation of TOR complex 1. Am J Physiol Endocrinol Metab. 2009;296:E592-602.
   
   
8. Hagan GN, Lin Y, Magnuson MA, Avruch J, Czech MP. A Rictor-Myo1c complex participates in dynamic cortical actin events in 3T3-L1 adipocytes. Mol Cell Biol. 2008;28:4215-26.
   
   
9. Praskova M, Xia F, Avruch J. MOBKL1A/MOBKL1B phosphorylation by MST1 and MST2 inhibits cell proliferation. Curr Biol. 2008;18:311-21.