CNK1 Is a Scaffold Protein That Regulates Src-mediated Raf-1 Activation

Ziogas, Algirdas; Moelling, Karin; Radziwill, Gerald

doi:10.1074/jbc.m413327200

Cited by 52 publications

(59 citation statements)

References 55 publications

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“…The following antibodies were used: anti-NF-κB1 p105/p50, anti-NF-κB2 p100/p52, anti-RelA, anti-RelB, anti-pS176/180(177/ 181) IKKα(β), anti-IKKα, anti-IKKβ, anti-pS32 IκBα, and anti-IκBα (Cell Signaling Technology); anti-pY204 ERK1/ 2, anti-ERK2, and anti-α-tubulin (Santa Cruz Biotechnology). Anti-CNK1 antibody was described elsewhere (11).…”

Section: Reagentsmentioning

confidence: 99%

“…Retroviral pMSCV vectors expressing shRNAs against CNK1 [shCNK1; 5′-GTAGTACTGCTAGTCATGG-3′; targeting the 3′ untranslated region (UTR)] or firefly luciferase (shGL2; 5′-CGTACGCGGAATACTTCGA-3′) and viral transduction were described previously (11). Transduced MDA-MB-231 and MCF10A cells were selected with 1 μg/mL puromycin to obtain stable polyclonal cell lines.…”

Section: Plasmidsmentioning

confidence: 99%

“…Previously, we described that CNK1 mediates vascular endothelial growth factor-dependent ERK signaling (11), and therefore we tested whether ERK signaling influences CNK1-driven cell invasion. MDA-MB-231 shControl and shCNK1 cells were subjected to Matrigel invasion assays in the presence or absence of the MEK inhibitor PD98059.…”

Section: Cnk1-mediated Invasion Depends On the Nf-κb Pathwaymentioning

confidence: 99%

“…However, CNK2 does not mediate epidermal growth factor-induced transient ERK activation required for proliferation (10). The ubiquitously expressed human CNK1 acts as a scaffold protein in Src-mediated Raf activation and allows vascular endothelial growth factor-dependent stimulation of the Raf-MEK-ERK pathway (11). Similar to CNK2, CNK1 is not required for epidermal growth factor-induced ERK activation (10).…”

Section: Introductionmentioning

confidence: 99%

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CNK1 Promotes Invasion of Cancer Cells through NF-κB–Dependent Signaling

Fritz

Radziwill

2010

Molecular Cancer Research

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Hallmarks of cancer cells are uncontrolled proliferation, evasion of apoptosis, angiogenesis, cell invasion, and metastasis, which are driven by oncogenic activation of signaling pathways. Herein, we identify the scaffold protein CNK1 as a mediator of oncogenic signaling that promotes invasion in human breast cancer and cervical cancer cells. Downregulation of CNK1 diminishes the invasiveness of cancer cells and correlates with reduced expression of matrix metalloproteinase 9 (MMP-9) and membrane-type 1 MMP (MT1-MMP). Ectopic expression of CNK1 elevates MT1-MMP promoter activity in a NF-κB-dependent manner. Moreover, CNK1 cooperates with the NF-κB pathway, but not with the extracellular signal-regulated protein kinase pathway, to promote cell invasion. Mechanistically, CNK1 regulates the alternative branch of the NF-κB pathway because knockdown of CNK1 interferes with processing of NF-κB2 p100 to p52 and its localization to the nucleus. In agreement with this, the invasion of CNK1-depleted cells is less sensitive to RelB downregulation compared with the invasion of control cells. Moreover, CNK1-dependent MT1-MMP promoter activation is blocked by RelB siRNA. Thus, CNK1 is an essential mediator of an oncogenic pathway involved in invasion of breast and cervical cancer cells and is therefore a putative target for cancer therapy. Mol Cancer Res; 8(3); 395-406. ©2010 AACR.

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Section: Reagentsmentioning

confidence: 99%

Section: Plasmidsmentioning

confidence: 99%

Section: Cnk1-mediated Invasion Depends On the Nf-κb Pathwaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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CNK1 Promotes Invasion of Cancer Cells through NF-κB–Dependent Signaling

Fritz

Radziwill

2010

Molecular Cancer Research

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“…Such proteins might also protect the Nregion against phosphatases and could provide an explanation for the constitutive S446 phosphorylation of B-Raf that is observed in different cell types and species. Indeed, recent findings have shown that the adapter protein CNK1 binds to Raf-1 in an inducible and Ras-GTP-dependent manner, whereas its association with B-Raf and pre-activated Raf-1 mutants with increased N-region charge is constitutive and Ras-GTP independent (Ziogas et al, 2005). Thus, proteins like CNK1 may assist in the stabilization of the primed state.…”

Section: Regulation Of B-raf Signallingmentioning

confidence: 99%

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

et al. 2006

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The BRAF V600E mutation is found in approximately 6% of human cancers and mimics the phosphorylation of the kinase domain activation segment. In wild-type B-Raf (B-Raf wt ), activation segment phosphorylation is thought to cooperate with negative charges within the N-region for full activation. In contrast to Raf-1, the N-region of B-Raf is constitutively negatively charged owing to the presence of residues D447/D448 and the phosphorylation of S446. Therefore, it has been suggested that this hallmark predisposes B-Raf for oncogenic activation. In this study, we demonstrate that neutralizing mutations of these residues (in particular S446 and S447), or uncoupling of B-Raf from Ras-guanine 5 0 -triphosphate (GTP), strongly reduce the biological activity of B-Raf in a PC12 cell differentiation assay. We also confirm that S365 is a 14-3-3 binding site, and determine that mutation of this residue rescues the impaired biological activity of B-Raf proteins with a neutralized N-region, suggesting that the N-region opposes a 14-3-3-mediated transition into an inactive conformation. However, in the case of B-Raf V600E , although complete N-region neutralization resulted in a 2.5-fold reduction in kinase activity in vitro, this oncoprotein strongly induced PC12 differentiation or transformation and epithelial-mesenchymal transition of MCF-10A cells regardless of its N-region charge. Furthermore, the biological activity of B-Raf V600E was independent of its ability to bind Ras-GTP. Our analysis identifies important regulatory differences between B-Raf wt and B-Raf V600E and suggests that B-Raf V600E cannot be inhibited by strategies aimed at blocking S446 phosphorylation or Ras activation. Keywords: Ras; 14-3-3 proteins; b-Catenin; E-cadherin; mammary epithelial cells IntroductionThe Ras/Raf/mitogen-activated/extracellular-regulated kinase (MEK)/extracellular signal regulated kinase (ERK) pathway plays a pivotal role in control of proliferation and differentiation and, owing to its role as a gatekeeper of this pathway, Raf appears an attractive therapeutic target (O'Neill and Kolch, 2004;Wilhelm et al., 2004). The Raf-kinase family comprises the A-Raf, B-Raf and Raf-1 isoforms in vertebrates as well as D-Raf and LIN-45 in Drosophila and Caenorhabditis, respectively. B-Raf, the major ERK activator in vertebrates, is required for the maintenance of basal ERK activity and displays the most potent transforming activity (Papin et al., 1998;Brummer et al., 2002;Mercer and Pritchard, 2003). All isoforms share three highly conserved regions (CRs; Figure 1a): the N-terminal CR1 contains the Ras-guanine 5 0 -triphosphate (GTP)-binding domain (RBD), which initiates the interaction with Ras-GTP through a conserved arginine residue (R188 in B-Raf) that is required for the recruitment and activation of Raf at the plasma membrane. Consequently, mutation of this residue prevents Ras/Raf interaction and renders D-Raf, B-Raf and Raf-1 unresponsive to most extracellular signals (Hou et al., 1995;Marais et al., 1997;Brummer et al., 2002). The ...

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Intracellular Signal Transduction Cascades

Matthews¹,

Gerritsen²

2010

Targeting Protein Kinases for Cancer Therapy

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CNK1 Is a Scaffold Protein That Regulates Src-mediated Raf-1 Activation

Cited by 52 publications

References 55 publications

CNK1 Promotes Invasion of Cancer Cells through NF-κB–Dependent Signaling

CNK1 Promotes Invasion of Cancer Cells through NF-κB–Dependent Signaling

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

Intracellular Signal Transduction Cascades

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