ABSTRACT
Mammalian target of rapamycin (mTOR) plays a range of crucial roles in cell survival, growth, proliferation, metabolism, and morphology. However, mTOR forms two distinct complexes, mTOR complex 1 and mTOR complex 2 (mTORC1 and mTORC2), via association with a series of different components; this allows the complexes to execute their wide range of functions. This study explores further the composition of the mTORC2 complex. Utilizing Rictor knock-out cells, immunoprecipitation and mass spectrometry, a novel Rictor associated protein, heterogeneous nuclear ribonucleoprotein M (hnRNP M), was identified. The association between hnRNP M and Rictor was verified using recombinant and endogenous protein and the binding site was found to be within aa 1~532 of hnRNP M. The presence of hnRNP M significantly affects phosphorylation of SGK1 S422, but not of Akt S473, PKCα S657 and PKCζ T560. Furthermore, hnRNP M also plays a critical role in muscle differentiation because knock-down of either hnRNP M or Rictor in C2C12 myoblasts reduced differentiation. This decrease is able to be rescued by overexpression SGK S422D in hnRNP M knockdown C2C12 myoblasts. Taken together, we have identified a novel Rictor/mTOR binding molecule, hnRNP M, that allows mTORC2 signaling to phosphorylate SGK1 thus regulating muscle differentiation.
Subject(s)
Heterogeneous-Nuclear Ribonucleoprotein Group M/metabolism , Myoblasts/cytology , Rapamycin-Insensitive Companion of mTOR Protein/genetics , Rapamycin-Insensitive Companion of mTOR Protein/metabolism , Animals , Binding Sites , Cell Differentiation , Cell Line , Gene Knockout Techniques , HEK293 Cells , Heterogeneous-Nuclear Ribonucleoprotein Group M/chemistry , Heterogeneous-Nuclear Ribonucleoprotein Group M/genetics , Humans , Immediate-Early Proteins/chemistry , Immediate-Early Proteins/metabolism , Mice , Myoblasts/metabolism , Phosphorylation , Protein Binding , Protein Kinase C-alpha/chemistry , Protein Kinase C-alpha/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Signal TransductionABSTRACT
Colorectal cancers with metastatic potential secrete the glycoprotein carcinoembryonic antigen (CEA). CEA has been implicated in colorectal cancer metastasis by inducing Kupffer cells to produce inflammatory cytokines which, in turn, make the hepatic micro-environment ideal for tumor cell implantation. CEA binds to the heterogeneous ribonucleoprotein M (hnRNP M) which acts as a cell surface receptor in Kupffer cells. The amino acid sequence in CEA, which binds the hnRNP M receptor, is Tyr-Pro-Glu-Leu-Pro-Lys. In this study, the structure of Ac-Tyr-Pro-Glu-Leu-Pro-Lys-NH2 (YPELPK) was investigated using electronic circular dichroism, vibrational circular dichroism, and molecular dynamics simulations. The binding of the peptide to hnRNP M was also investigated using molecular docking calculations. The biological activity of YPELPK was studied using differentiated human THP-1 cells, which express hnRNP M on their surface and secrete IL-6 when stimulated by CEA. YPELPK forms a stable polyproline-II helix and stimulates IL-6 production of THP-1 cells at micromolar concentrations.
Subject(s)
Carcinoembryonic Antigen/chemistry , Heterogeneous-Nuclear Ribonucleoprotein Group M/agonists , Peptide Fragments/chemistry , Amino Acid Sequence , Amino Acid Substitution , Carcinoembryonic Antigen/pharmacology , Cell Line , Heterogeneous-Nuclear Ribonucleoprotein Group M/chemistry , Heterogeneous-Nuclear Ribonucleoprotein Group M/metabolism , Humans , Hydrogen Bonding , Interleukin-6/biosynthesis , Molecular Dynamics Simulation , Peptide Fragments/pharmacology , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Surface Properties , ThermodynamicsABSTRACT
Heterogeneous nuclear ribonucleoprotein-M (hnRNP-M) is an abundant nuclear protein that binds to pre-mRNA and is a component of the spliceosome complex. A direct interaction was detected in vivo between hnRNP-M and the human spliceosome proteins cell division cycle 5-like (CDC5L) and pleiotropic regulator 1 (PLRG1) that was inhibited during the heat-shock stress response. A central region in hnRNP-M is required for interaction with CDC5L/PLRG1. hnRNP-M affects both 5' and 3' alternative splice site choices, and an hnRNP-M mutant lacking the CDC5L/PLRG1 interaction domain is unable to modulate alternative splicing of an adeno-E1A mini-gene substrate.
