Direct Interaction of the N-terminal Domain of Focal Adhesion Kinase with the N-terminal Transactivation Domain of p53

Abstract

Focal adhesion kinase (FAK) is a nonreceptor kinase that is overexpressed in many types of tumors and associates with multiple cell surface receptors and intracellular signaling proteins through which it can play an important role in survival signaling. A link between FAK and p53 in survival signaling has been reported, although the molecular basis of these events has not been described. In the present study, we report that FAK physically and specifically interacts with p53 as demonstrated by pull-down, immunoprecipitation, and co-localization analyses. Using different constructs of N-terminal, central, and C-terminal fragments of FAK and p53 proteins, we determined that the N-terminal fragment of FAK directly interacts with the N-terminal transactivation domain of p53. Inhibition of p53 with small interfering p53 RNA resulted in a decreased complex of FAK and p53 proteins in 293 cells, and induction of p53 with doxorubicin in normal human fibroblasts caused an increase of FAK and p53 interaction. Introduction of the FAK plasmid into p53-null SAOS-2 cells was able to rescue these cells from apoptosis induced by expression of wild type p53. In HCT 116 colon cancer cells, co-transfection of FAK plasmid with p21, MDM-2, and BAX luciferase plasmids resulted in significant inhibition of p53-responsive luciferase activities, demonstrating that FAK can reduce transcriptional activity of p53. The results of the FAK and p53 interaction study strongly support the conclusion that FAK can suppress p53-mediated apoptosis and inhibit transcriptional activity of p53. This provides a novel mechanism for FAKp53-mediated survival/apoptotic signaling. The focal adhesion kinase (FAK)1 is a nonreceptor cytoplasmic 125-kDa protein-tyrosine kinase that controls a number of cellular signaling pathways, including proliferation, cell spreading, motility, and survival (1-4). The FAK gene was first isolated from chicken (1) and mouse (5) fibroblasts and then from human T and B lymphocytes (6) and human sarcoma samples (7). The FAK gene is located on chromosome 8q24 (8, 9) in humans and on chromosome 15 in mice (8).FAK is overexpressed in many types of tumors (7, 10 -14), and it was suggested that FAK up-regulation occurred in early stages of tumorigenesis (13,15). FAK mRNA levels have been shown to be increased in premalignant adenomatous tissues and invasive and metastatic tumors (7, 15). Increased FAK mRNA expression was demonstrated in adenomatous tissues, invasive tumors, and metastatic tumors (7, 16). Recently, real time PCR analysis of matched samples of normal colon mucosa, colorectal carcinoma, and liver metastases demonstrated increased FAK mRNA and protein levels in tumor and metastatic tissues versus normal tissues (16). Cloning and characterization of the FAK promoter demonstrated NF-B and p53 transcription factor binding sites that had potential importance for regulation of FAK transcription (17).FAK was originally isolated as a tyrosine phosphorylated protein in v-Src-transformed chicken embryo fibroblasts (1, 18). The struct...

Mammary Epithelial-Specific Disruption of Focal Adhesion Kinase Retards Tumor Formation and Metastasis in a Transgenic Mouse Model of Human Breast Cancer

“…p53 is a known tumor suppressor and regulator of both the G 1 /S and G 2 /M transitions, 85 as well as a transcriptional repressor of cyclin B. [85][86][87] Interestingly, FAK has been recently reported to bind directly to p53 and reduce its transcriptional activity 41 and proliferation of FAK Ϫ/Ϫ embryonic fibroblasts requires inactivation of p53, 88 whereas FAK-interacting protein (FIP200) can bind p53 and extend its half-life. 89 As discussed by Mitra and Schlaepfer, 15 because p53 and FAK may compete for the same binding region on FIP200, conditions of limited FAK levels may promote FIP200 stabilization and enhancement of p53 activity.…”

“…31,32 In addition, FAK is known to modulate the activity of several transcription factors important in human breast cancer, such as NF-B, CREB, STAT1, KLF8, Smad1, Prx1, AP-1, and p53. [33][34][35][36][37][38][39][40][41] Hence, FAK directly regulates many fundamental adhesion and growth factor signaling pathways and transcription factors central in human cancer, and is therefore well poised to regulate mammary tumorigenesis and metastasis.…”

Adenoviruses increase endothelial cell proliferation, migration, and tube formation: Partial reversal by the focal adhesion kinase inhibitor, FRNK

“…As such, FAK-mediated signal transduction is extremely complex with FAK modulating cell survival via p53-dependent and p53-independent means (GabarraNieko et al, 2003). FAK mediates p-53 independent cell survival through the PI3K/Akt, MAPK/Erk and Jun pathways (Gabarra-Nieko et al, 2003), whereas FAK modulates p53-dependent cell survival by binding to and reducing the transcriptional activity of p53 (Golubovskaya, et al, 2005). As such, ectopic expression of FRNK in endothelial cells may oppose adenoviral-mediated PI3K/AKT activation while increasing the transcriptional activation of p53, which would have a pro-apoptotic effect.…”

Pyk2 Inhibition of p53 as an Adaptive and Intrinsic Mechanism Facilitating Cell Proliferation and Survival

“…Nuclear-targeted FAK promotes p53 turnover by facilitating Mdm2-dependent ubiquitination of p53 (10). FAK regulation of p53 levels is dependent upon the FAK FERM domain but independent of FAK kinase activity (13,14). p53 is a transcription factor that regulates many targets such as the p21Cip/ WAF1 (p21) cyclin-dependent kinase inhibitor (15,16).…”