RACK1 inhibits the serum‐ and anchorage‐independent growth of v‐Src transformed cells

Mamidipudi, Vidya; Chang, Betty; Harte, Rachel A.; Lee, Kelly C.; Cartwright, Christine A.

doi:10.1016/j.febslet.2004.03.125

Cited by 23 publications

(25 citation statements)

References 37 publications

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“…For example, the inhibitory effect of the peptide that enhances the βIIPKC-RACK1-Src interaction on: a) tyrosine phosphorylation of the Src-specific substrates Sam68 and Stat3 (Fig 1), b) induction of the Src effectors Myc and cyclin D1 (Fig 1), and c) on Src-mediated p190RhoGAP activity and actin-cytoskeleton rearrangement [ 9 ], is strikingly similar to the effect observed with: a) the Src-specific tyrosine kinase inhibitors SU6656, PP2 or PP1 [reviewed in 23,30 ], b) dominant-negative mutants of Src [ 23,30 ], or c) overexpression of the endogenous Src inhibitor RACK1 [ 6 ]. Conversely, the activating effects of the peptides that disrupt the βIIPKC-RACK1-Src interaction are similar to those observed with v-Src [ 23,30,31 ], constitutively-active Y527F c-Src [ 23,30 ] or with depletion of RACK1 [ 6 ]. Thus, perturbing RACK1's interaction with Src is a major mechanism by which the peptides function to regulate cell growth.…”

Section: Discussionsupporting

confidence: 52%

Peptide modulators of Src activity in G1 regulate entry into S phase and proliferation of NIH 3T3 cells

Mamidipudi

Miller

Mochly‐Rosen

et al. 2007

Biochemical and Biophysical Research Communications

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Cascades of kinases and phosphatases are regulated by selective protein-protein interactions that are essential for signal transduction. Peptide modulators of these interactions have been used to dissect the function of individual components of the signaling cascade, without relying on either the overor underexpression of proteins. Previously, we identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src tyrosine kinases. Here we utilized cell-permeable peptides that selectively disrupt or enhance the interaction of RACK1 and Src to further examine the function of RACK1. Our results provide direct physiologic evidence that RACK1 regulates growth of NIH3T3 cells by suppressing the activity of Src and other cell cycle regulators in G 1 , and delaying entry into S phase. They also demonstrate the potential for using peptide modulators of Src activity as a tool for regulating cell growth, and for designing new strategies for cancer therapy that target specific protein-protein interactions. KeywordsSrc; RACK1; PKC; tyrosine kinases; G 1 /S transition; cell cycle regulation RACK1 is the founding member of a family of intracellular receptors for activated C kinase (PKC) collectively called RACKs [reviewed in 1 ]. RACKs determine the specificity and function of PKC isozymes by selectively anchoring activated isozymes to specific subcellular sites, and near to specific substrates that mediate distinct cellular functions. For example, εRACK anchors activated εPKC, which protects the heart from ischemic and reperfusion damage, whereas RACK1 anchors activated βIIPKC, which mediates phenylepherine-induced cardiac hypertrophy [reviewed in 2 ]. *Corresponding author: M211 Alway Building, 300 Pasteur Drive, Stanford University School of Medicine, Stanford,, e-mail: chris.cartwright@stanford.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errorsmaybe discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Materials and methods Cell Culture and Cell Proliferation AssaysNIH3T3 cells were maintained in Dulbecco's modified Eagle medium (Mediatech, Herndon, VA). Cells were supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT), unless otherwise stated. Methods for peptide incubations were previously described [ 9 ]. For cell proliferation assays, cells were seeded in 96-well plates (5 × 10 3 cells/well). Peptides and 10% Alamar Blue were added and absorbance was measured hourly for 8 h at a wavelength of 562 and 595 nm using a Vmax kinetic microplate reader (Molecular Devices, Palo Alto, CA). Fresh peptide was added every 2 h. Reduction of alamarBlue was calculated from absorbance, according to t...

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Section: Discussionsupporting

confidence: 52%

Peptide modulators of Src activity in G1 regulate entry into S phase and proliferation of NIH 3T3 cells

Mamidipudi

Miller

Mochly‐Rosen

et al. 2007

Biochemical and Biophysical Research Communications

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“…Moreover, upon PKC activation, RACK1, the founding member of the family of receptors for activated C kinase, colocalises with Src at the plasma membrane and functions as a substrate, binding partner and inhibitor of Src (Chang et al, 2002). Among those proteins, both AFAP-110 and RACK1 have been shown to be involved in the regulation of podosome formation (Gatesman et al, 2004;Mamidipudi et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

A signalling cascade involving PKC, Src and Cdc42 regulates podosome assembly in cultured endothelial cells in response to phorbol ester

Tatin

Varon

Génot

et al. 2006

Journal of Cell Science

151

137

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The involvement of Src, Cdc42, RhoA and PKC in the regulation of podosome assembly has been identified in various cell models. In endothelial cells, the ectopic expression of constitutively active mutants of Src or Cdc42, but not RhoA, induced the formation of podosomes. Shortterm exposure to phorbol-12-myristate-13-acetate (PMA) induced the appearance of podosomes and rosettes after initial disruption of stress fibres. Molecular analysis of PMA-induced podosomes and rosettes revealed that their composition was identical to that of podosomes described in other models. Pharmacological inhibition and siRNA knock-down experiments revealed that both PKC␣ ␣ and PKC␦ ␦ isotypes were necessary for podosome assembly. However, only constitutively active PKC␣ ␣ could mimic PMA in podosome formation. Src, Cdc42 and RhoA were required downstream of PKCs in this process. Src could be positioned between PKC and Cdc42 in a linear cascade leading to podosome assembly. Using in vitro matrix degradation assays, we demonstrated that PMA-induced podosomes are endowed with proteolytic activities involving MT1-MMP-mediated activation of MMP2. Endothelial podosomes may be involved in subendothelial matrix degradation during endothelium remodelling in pathophysiological processes.

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“…RACK1 is known to regulate integrin-mediated cell adhesion (Buensuceso et al, 2001;Hermanto et al, 2002;Kiely et al, 2002Kiely et al, , 2005Cox et al, 2003;Mamidipudi et al, 2004b;Vomastek et al, 2007). Moreover, RACK1 acts as a scaffolding protein to integrate adhesion and growth factor signaling necessary for cell migration.…”

Section: Discussionmentioning

confidence: 99%

A novel pro-apoptotic function of RACK1: suppression of Src activity in the intrinsic and Akt pathways

Mamidipudi

Cartwright

2009

Oncogene

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Earlier we showed that RACK1 regulates growth of human colon cells by suppressing Src activity at G 1 and mitotic checkpoints. Here, we show that RACK1 also induces apoptosis of the cells, partly by inhibiting Src. In the intrinsic pathway, RACK1 inhibits expression of anti-apoptotic Bcl-2 and Bcl-X L , induces expression of proapoptotic Bim, targets Bim and Bax to the mitochondria, induces oligomerization of Bax (which requires Bim and inhibition of Src), depolarizes mitochondria membranes, releases cytochrome c, and activates caspases-9 and -3 and death substrates. Bax and Bim are required for RACK1-mediated mitochondrial cell death. RACK1-induced oligomerization of Bax is required for staurosporine-mediated cell death. RACK1 also induces apoptosis by blocking Src activation of the Akt cell survival pathway. This leads to activation of the transcription factor FOXO3, a potent inducer of apoptosis and G 1 arrest. Collectively, our results show that RACK1, partly by inhibiting Src, promotes mitochondrial cell death and blocks Akt-mediated cell survival. Thus, RACK1 inhibits growth and induces death of colon cells. Exploitation of these dual functions could lead to novel colon cancer therapies that mimic RACK1 function.

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RACK1 inhibits the serum‐ and anchorage‐independent growth of v‐Src transformed cells

Cited by 23 publications

References 37 publications

Peptide modulators of Src activity in G1 regulate entry into S phase and proliferation of NIH 3T3 cells

Peptide modulators of Src activity in G1 regulate entry into S phase and proliferation of NIH 3T3 cells

A signalling cascade involving PKC, Src and Cdc42 regulates podosome assembly in cultured endothelial cells in response to phorbol ester

A novel pro-apoptotic function of RACK1: suppression of Src activity in the intrinsic and Akt pathways

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