Isolation of mammalian Lin-2A, a protein that interacts with a highly conserved region of Myc proteins

Abstract

A cell responds to the extracellular environment by transducing signals from the cell surface to the nucleus. This results in chances that eventually affect gene expression. This process is especially important during embryonic development, which requires cell-cell communication. Members of the Myc-family (c-Myc, N-Myc, and L-Myc) are nuclear phosphoproteins implicated in the control of cell growth and differentiation. Accumulating biochemical, structural and genetic evidence affords the view that the function of the Myc-family proteins in these diverse processes is in part due to their role as sequence-specific transcription factors. The correlation between Myc induction and activation in response to growth factors indicates that Myc is a target of signal transduction cascades.;To identify proteins that interact with the Myc proteins, a yeast two-hybrid approach was employed utilizing the highly conserved Myc Box 1(MB1) region of the transactivational domain of c-Myc. A novel human protein, as well as its murine homologue, termed hLin-2A was identified that interacted specifically with the MB1 region. Significant homology exists between this Myc-interacting protein and members of membrane-associated guanylate kinase (MAGUK) family of proteins, which are implicated in cell-cell contact and intracellular signaling. It is demonstrated in vitro and in vivo that Myc and hLin-2A associate and the regions of interaction are defined within both proteins. RNA expression studies, as revealed by Northern blotting and in situ hybridization analyses, implicate a role in neuronal and kidney development.;hLin-2A is a homologue of the LIN-2A protein in C.elegans, which is required for the LET-23 receptor tyrosine kinase(RTK)/LET-60 Ras signaling pathway during vulval development. Since MAGUK proteins are known to interact with transmembrane proteins, we tested the interaction of hLin-2A with EPH-family RTKs, proteins known to localize in regions of cell-cell contact during development. Over-expression studies in COS7 cells demonstrate association of hLin-2A with several EPH-family RTKs. Additional binding assays are presented that study these associations.;In summary, this thesis study describes the isolation and characterization of hLin-2A, a novel Myc-interacting protein. hLin-2A might provide insight into the understanding of how MAGUK proteins function intracellularly through interaction with transcription factors that eventually affect the transcription of genes.