Nuclear FAK Promotes Cell Proliferation and Survival through FERM-Enhanced p53 Degradation

Department of Reproductive Medicine, Moores Cancer Center, University of California, San Diego, 3855 Health Sciences Drive, MC0803, La Jolla, CA 92093, USA.
Molecular Cell (Impact Factor: 14.02). 02/2008; 29(1):9-22. DOI: 10.1016/j.molcel.2007.11.031
Source: PubMed


FAK is known as an integrin- and growth factor-associated tyrosine kinase promoting cell motility. Here we show that, during mouse development, FAK inactivation results in p53- and p21-dependent mesodermal cell growth arrest. Reconstitution of primary FAK-/-p21-/- fibroblasts revealed that FAK, in a kinase-independent manner, facilitates p53 turnover via enhanced Mdm2-dependent p53 ubiquitination. p53 inactivation by FAK required FAK FERM F1 lobe binding to p53, FERM F2 lobe-mediated nuclear localization, and FERM F3 lobe for connections to Mdm2 and proteasomal degradation. Staurosporine or loss of cell adhesion enhanced FERM-dependent FAK nuclear accumulation. In primary human cells, FAK knockdown raised p53-p21 levels and slowed cell proliferation but did not cause apoptosis. Notably, FAK knockdown plus cisplatin triggered p53-dependent cell apoptosis, which was rescued by either full-length FAK or FAK FERM re-expression. These studies define a scaffolding role for nuclear FAK in facilitating cell survival through enhanced p53 degradation under conditions of cellular stress.

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Available from: Ssang-Taek Steve Lim, Apr 08, 2015
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    • "To ultimately validate that it is the kinase activity that mediates FAK/PYK2 regulation of Wnt/β-catenin signaling, we examined GSK3β Y216 phosphorylation-dependent activation of Wnt/β-catenin signaling in FAK kinase-deficient MEFs (FAK R454/R454 knockin MEFs). A knock-in point mutation of lysine 454 to arginine within the catalytic domain inactivates FAK kinases activity (Lim et al., 2010) (Figure 4B, represented by abolished phosphorylation of FAK Y397 ) but leaves FAK's scaffolding activity intact (Lim et al., 2008). Consistent with our finding that FAK and PYK2 function redundantly in phosphorylating GSK3, despite of suppressed FAK activity in FAK R454/R454 MEFs, KD of PYK2 was required to inhibit GSK3β Y216 phosphorylation. "
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    ABSTRACT: Aberrant activation of Wnt/β-catenin signaling plays an unequivocal role in colorectal cancer, but identification of effective Wnt inhibitors for use in cancer remains a tremendous challenge. New insights into the regulation of this pathway could reveal new therapeutic point of intervention, therefore are greatly needed. Here we report a novel FAK/PYK2/GSK3βY(216)/β-catenin regulation axis: FAK and PYK2, elevated in adenomas in APC(min/+) mice and in human colorectal cancer tissues, functioned redundantly to promote the Wnt/β-catenin pathway by phosphorylating GSK3β(Y216) to reinforce pathway output-β-catenin accumulation and intestinal tumorigenesis. We previously showed that Wnt-induced β-catenin accumulation requires Wntinduced GSK3β/β-TrCP interaction; the current study revealed that phosphorylation of GSK3β(Y216) was a molecular determinant of GSK3β recruitment of β-TrCP. Pharmacological inhibition of FAK/PYK2 suppressed adenoma formation in APC(min/+) mice accompanied with reduced intestinal levels of phospho-SK3β(Y216) and β-catenin, indicating that FAK/PYK2/GSK3β(Y216) axis is critical for the activation of Wnt/β-catenin signaling in APCdriven intestinal tumorigenesis.
    eLife Sciences 08/2015; 4. DOI:10.7554/eLife.10072 · 9.32 Impact Factor
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    • "An OFSS-induced nucleocytoplasmic shuttling mechanism for Pyk2 has yet to be described, but other studies in fibroblasts have observed Pyk2’s nucleocytoplasmic shuttling behavior in response to membrane depolarization [49],[50]. Nucleocytoplasmic shuttling of Pyk2 is possible through the nuclear localization sequence (NLS) and the nuclear export sequence (NES) that are both located in the FERM domain [51]–[53]. The changes observed in Cox-2 expression and osteopontin transcription might be mediated by an OFSS-induced association of Pyk2 and MBD2. "
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    ABSTRACT: Mechanical stimulation of the skeleton promotes bone gain and suppresses bone loss, ultimately resulting in improved bone strength and fracture resistance. The molecular mechanisms directing anabolic and/or anti-catabolic actions on the skeleton during loading are not fully understood. Identifying molecular mechanisms of mechanotransduction (MTD) signaling cascades could identify new therapeutic targets. Most research into MTD mechanisms is typically focused on understanding the signaling pathways that stimulate new bone formation in response to load. However, we investigated the structural, signaling and transcriptional molecules that suppress the stimulatory effects of loading. The high bone mass phenotype of mice with global deletion of either Pyk2 or Src suggests a role for these tyrosine kinases in repression of bone formation. We used fluid shear stress as a MTD stimulus to identify a novel Pyk2/Src-mediated MTD pathway that represses mechanically-induced bone formation. Our results suggest Pyk2 and Src function as molecular switches that inhibit MTD in our mechanically stimulated osteocyte culture experiments. Once activated by oscillatory fluid shear stress (OFSS), Pyk2 and Src translocate to and accumulate in the nucleus, where they associate with a protein involved in DNA methylation and the interpretation of DNA methylation patterns -methyl-CpG-binding domain protein 2 (MBD2). OFSS-induced Cox-2 and osteopontin expression was enhanced in Pyk2 KO osteoblasts, while inhibition of Src enhanced osteocalcin expression in response to OFSS. We found that Src kinase activity increased in the nucleus of osteocytes in response to OFSS and an interaction activated between Src (Y418) and Pyk2 (Y402) increased in response to OFSS. Thus, as a mechanism to prevent an over-reaction to physical stimulation, mechanical loading may induce the formation of a Src/Pyk2/MBD2 complex in the nucleus that functions to suppress anabolic gene expression.
    PLoS ONE 05/2014; 9(5):e97942. DOI:10.1371/journal.pone.0097942 · 3.23 Impact Factor
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    • "Cells were propagated adherently on plastic or replated on lowbinding poly 2-hydroxyethyl methacrylate (poly-HEMA) coated plates for experimental anchorage-independent analyses. The coding sequence for fluorescent mCherry protein (pmCherry-C1, Clontech) was subcloned into the lentiviral expression vector (pCDH-CMV- MSC1, System Biosciences) and recombinant lentivirus produced as described [29]. Lentivirus-transduced mCherry-expressing 5009- MOVCAR, shRNA merlin, and shRNA Scr (control) cells were selected by growth in puromycin (2 μg/ml), expanded, and frozen as low passage stocks. "
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    ABSTRACT: Focal adhesion kinase (FAK) is overexpressed in serous ovarian cancer. Loss of merlin, a product of the neurofibromatosis 2 tumor suppressor gene, is being evaluated as a biomarker for FAK inhibitor sensitivity in mesothelioma. Connections between merlin and FAK in ovarian cancer remain undefined. Nine human and two murine ovarian cancer cell lines were analyzed for growth in the presence of a small molecule FAK inhibitor (PF-271, 0.1 to 1μM) for 72h. Merlin was evaluated by immunoblotting and immunostaining of a human ovarian tumor tissue array. Growth of cells was analyzed in an orthotopic tumor model and evaluated in vitro after stable shRNA-mediated merlin knockdown. Greater than 50% inhibition of OVCAR8, HEY and ID8-IP ovarian carcinoma cell growth occurred with 0.1μM PF-271 in anchorage-independent (p<0.001) but not in adherent culture conditions. PF-271-mediated reduction in FAK Y397 phosphorylation occurred independently of growth inhibition. Suspended growth of OVCAR3, OVCAR10, IGROV1, IGROV1-IP, SKOV3, SKOV3-IP, A2780, and 5009-MOVCAR was not affected by 0.1μM PF-271. Merlin expression did not correlate with serous ovarian tumor grade or stage. PF-271 (30mg/kg, BID) did not inhibit 5009-MOVCAR tumor growth and merlin knockdown in SKOV3-IP and OVCAR10 cells did not alter suspended cell growth upon PF-271 addition. Differential responsiveness to FAK inhibitor treatment were observed. Intrinsic low merlin protein levels correlated with PF-271-mediated anchorage-independent growth inhibition, but reduction in merlin expression did not induce sensitivity to FAK inhibition. Merlin levels may be useful for patient stratification in FAK inhibitor trials.
    Gynecologic Oncology 04/2014; 134(1). DOI:10.1016/j.ygyno.2014.04.044 · 3.77 Impact Factor
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