RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, inhibits activity of src tyrosine kinases and growth of NIH 3T3 cells

Mol Cell Biol. 1998 Jun;18(6):3245-56. doi: 10.1128/MCB.18.6.3245.

Abstract

To isolate and characterize proteins that interact with the unique domain and SH3 and SH2 domains of Src and potentially regulate Src activity, we used the yeast two-hybrid assay to screen a human lung fibroblast cDNA library. We identified RACK1, a receptor for activated C kinase and a homolog of the beta subunit of G proteins, as a Src-binding protein. Using GST-Src fusion proteins, we determined that RACK1 binds to the SH2 domain of Src. Coimmunoprecipitation of Src and RACK1 was demonstrated with NIH 3T3 cells. Purified GST-RACK1 inhibited the in vitro kinase activity of Src in a concentration-dependent manner. GST-RACK1 (2 microM) inhibited the activities of purified Src and Lck tyrosine kinases by 40 to 50% but did not inhibit the activities of three serine/threonine kinases that we tested. Tyrosine phosphorylation on many cellular proteins decreased in 293T cells that transiently overexpressed RACK1. Src activity and cell growth rates decreased by 40 to 50% in NIH 3T3 cells that stably overexpressed RACK1. Flow cytometric analyses revealed that RACK1-overexpressing cells do not show an increased rate of necrosis or apoptosis but do spend significantly more time in G0/G1 than do wild-type cells. Prolongation of G0/G1 could account for the increased doubling time of RACK1-overexpressing cells. We suggest that RACK1 exerts its effect on the NIH 3T3 cell cycle in part by inhibiting Src activity.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Animals
  • Cell Division
  • Enzyme Activation
  • G1 Phase
  • GTP-Binding Proteins / metabolism*
  • Humans
  • Mice
  • Peptides / metabolism*
  • Phosphorylation
  • Protein Kinase C / metabolism*
  • Receptors for Activated C Kinase
  • Resting Phase, Cell Cycle
  • Tyrosine / metabolism
  • src Homology Domains
  • src-Family Kinases / metabolism*

Substances

  • Peptides
  • Receptors for Activated C Kinase
  • peptide I
  • Tyrosine
  • src-Family Kinases
  • Protein Kinase C
  • GTP-Binding Proteins