GFP-FRNK disrupts focal adhesions and induces anoikis in neonatal rat ventricular myocytes.
Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in adhesion-dependent signal transduction. FAK is highly expressed in cultured neonatal rat ventricular myocytes (NRVMs) and undergoes tyrosine autophosphorylation in response to cell adhesion, stretch, and growth factor stimulation. We previously showed that inhibition of FAK phosphorylation by adenovirally mediated overexpression of FRNK (the autonomously expressed C-terminal domain of FAK) prevented endothelin-1 (ET)-induced NRVM hypertrophy. One question raised by these studies was whether FRNK localized to focal adhesions and displaced FAK from sites required for downstream signaling. Therefore, we constructed a replication-defective adenovirus encoding a GFP-FRNK fusion protein (Adv-GFP-FRNK) and examined its effects on NRVM cytoarchitecture and signaling. Uninfected NRVMs contained small amounts of endogenous FRNK. NRVMs infected with Adv-GFP-FRNK expressed much larger amounts of a 66-/68-kDa protein that localized to costameres and focal adhesions. GFP-FRNK overexpression suppressed basal and ET-induced FAK phosphorylation and also inhibited ET-induced phosphorylation of PYK2, the other member of the FAK family of nonreceptor protein tyrosine kinases. In contrast, GFP-FRNK overexpression did not prevent ET-induced ERK, JNK, or p70S6K phosphorylation. Furthermore, GFP-FRNK resulted in the loss of detectable FAK and paxillin in focal adhesions, which was accompanied by reduced levels of total paxillin and, ultimately, cell detachment and apoptosis. We conclude that FRNK functions as a dominant-negative inhibitor of adhesion-dependent signaling by displacing FAK from focal adhesions and interfering with the anchorage of NRVMs that is necessary for cell survival, a process known as anoikis.
Heidkamp
Maria C
MC
Cardiovascular Institute, Loyola University Chicago, Stritch School of Medicine, Maywood, Ill, USA. mheidka@lumc.edu
Bayer
Allison L
AL
Kalina
Jared A
JA
Eble
Diane M
DM
Samarel
Allen M
AM
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D023102:Anoikis; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000378:metabolism; D016232:Endothelins / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions / Q000201:enzymology*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006352:Heart Ventricles / Q000737:chemistry / Q000166:cytology / Q000201:enzymology*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D051419:Paxillin; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000032:analysis; D015398:Signal Transduction
D003598:Cytoskeletal Proteins; D016232:Endothelins; D008164:Luminescent Proteins; D051419:Paxillin; D010750:Phosphoproteins; C496063:Pxn protein, rat; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C113183:FAK-related nonkinase; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat