Molecular Players: We focus on FAK and FAK binding partners because numerous studies have indicated that activation of this non-receptor protein tyrosine kinase within focal adhesions is central to the integrin-dependant signaling pathways that control cell growth, differentiation, and motility and nearly two thirds of the known integrin heterodimers including all b1, b3 or b5-containing integrins couple to FAK. Germline deletion of FAK in mice revealed that FAK is necessary for development beyond mid-gestation. Our laboratory was the first to show that cardiac- and/or smooth muscle restricted deletion of FAK mirrors several congenital cardiovascular diseases.
FAK: The association of FAK with integrins, results in activation of FAK kinase activity, autophosphorylation of Tyr397 and concomitant recruitment and phosphorylation of a number of additional adapter proteins (e.g. phosphoinositide 3-kinase, Crk associated substrate (CAS), paxillin, leupaxin, and GRB2) and/or GTPase regulators (e.g. GRAF, ASAP1) to the focal adhesion complex that leads to activation of various Map Kinase or G protein signaling cascades. The large repertoire of signaling proteins present in focal adhesions underscores the importance of these structures in propagating down-stream signals.
FRNK: We found that FAK activity is regulated in a unique fashion in smooth muscle cells and cardiomyocytes by the transient and specific expression of an endogenous inhibitor termed FAK-Related Non-Kinase (FRNK) which co-localizes with FAK in focal adhesions and serves to “buffer” FAK signaling in a time and tissue-specific fashion. Our studies indicate an essential role for FRNK in regulating smooth muscle cell maturation and restenosis following vascular injury. We are currently exploring the capacity of endogenous FRNK to modulate neonatal cardiomyocyte cell cycle withdrawal, since mis-expression of FRNK in the embryonic heart led to a non-compaction phenotype associated with impaired cardiomyocyte proliferation.
FRNK is expressed as a separate 41/43kDa protein transcribed from the FAK locus. Interestingly, FRNK expression is regulated by a promoter embedded within a FAK intron and a 6kb fragment of intronic sequence upstream of the FRNK ATG was sufficient to drive FRNK expression in vitro and in vivo. FRNK transcription initiates from a non-coding exon located approximately 300 bp upstream of the FRNK translational start site, and importantly, this exon is not contained in the FAK cDNA. FRNK localizes to focal adhesions and interacts with many of the aforementioned FAK C-terminal binding partners. Initial experiments in chicken embryo fibroblasts demonstrated that ectopic expression of FRNK resulted in the attenuation of matrix-stimulated FAK activation and inhibition of the rate of cell spreading, effects that could be rescued by overexpression of FAK or Src. Since FAK is probably activated by a homodimerization and transphosphorylation mechanism, FRNK likely inhibits FAK activity by forming inactive FRNK:FAK heterodimers.
Binding Partners: Over the past few years we have also identified novel specific binding partners for FAK in vascular smooth muscle, skeletal muscle, and heart and we continue to analyze whether these proteins interact with FAK in cells using conventional co-localization and immunoprecipitation assays, to define precise interaction sites using molecular approaches, and to define specific cellular functions using a variety of cell biology and mouse genetic approaches. We found that one such protein termed leupaxin regulates the toggling between smooth muscle cells motility and differentiation by forming protein complexes at the leading edge of cells or in nucleus that are essential for these functions. Other projects focus on the binding partner, GRAF, which we identified as a Rho-GAP that is essential for development and maintenance of skeletal muscle. Our goal is to determine when, where, and how these proteins function to control the complex morphogenetic changes observed during heart, skeletal muscle or vascular development or following a pathological insult with the hope that our studies will eventually lead to new molecular targets for the therapy of several devastating diseases.