RACK1 regulates Src-mediated Sam68 and p190RhoGAP signaling

Oncogene (2004) 23, 56825686

& 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00www.nature.com/oncRACK1 regulates Src-mediated Sam68 and p190RhoGAP signalingLaura D Miller1, Kelly C Lee1, Daria Mochly-Rosen2 and Christine A Cartwright*,11Department of Medicine, Stanford University, Stanford, CA 94305, USA; 2Department of Molecular Pharmacology, Stanford

University, Stanford, CA 94305, USARACK1 is the founding member of a familyof receptors

for activated C kinase collectivelycalled RACKs. Upon

activation of PKC, RACK1 co-localizes with the Src

tyrosine kinase at the plasma membrane and functions as

a substrate, binding partner and inhibitor of Src (as

measured in vitro), and a growth inhibitor in NIH 3T3

cells. To further analyze the function of RACK1 in Src

and PKC signaling, we utilized cell-permeable peptides

that modulate the interaction of RACK1 and bIIPKC,

therebyaffecting bIIPKC translocation and function. We

found that the association of bIIPKC and RACK1 is

necessaryfor Src phosphorylation of RACK1. Src activity

is required for tyrosine phosphorylation of RACK1, and

for RACK1 binding to Src, but not to bIIPKC.

Endogenous Src kinase activity, as measured by phosphorylation of Sam68 (a mitotic-specic Src substrate

involved in cell cycle regulation and RNA splicing) or

p190RhoGAP (a Src substrate and GTPase-activating

protein involved in actin reorganization), increases with

disruption of the Src-RACK1 complex, and decreases with

enhanced complex formation. RACK1 inhibits Srcmediated p190RhoGAP signaling and actin cytoskeleton

rearrangement. Thus, RACK1 functions as an endogenous

inhibitor of the Src kinase in diverse signaling pathways

that regulate distinct cellular functions. Our results

demonstrate the potential for using peptide modulators

of Src activityas a tool for uncovering the function of Src

in cells.Oncogene (2004) 23, 56825686. doi:10.1038/sj.onc.1207735

Published online 7 June 2004Keywords: Src; tyrosine kinase; RACK1; PKC; Sam68;

p190RhoGAPWhen regulated, the Src tyrosine kinase participates in a

wide array of signaling pathways that control cell

proliferation, differentiation, adhesion and survival

(reviewed in Thomas and Brugge, 1997). Deregulated

Src is oncogenic (Cartwright et al., 1987; Kmiecik and

Shalloway, 1987; Piwnica-Worms et al., 1987; Reynoldset al., 1987). Therefore, it is important to identify

mechanisms that regulate Src. In doing so, we will learn

how normal cells regulate their growth.PKC is a family of serine/threonine kinases (reviewed

in Newton, 1995). Upon activation, PKC isozymes

translocate to different subcellular sites where they

phosphorylate specic substrates that mediate distinct

cellular functions....