Identification of integrin-stimulated sites of serine phosphorylation in FRNK, the separately expressed C-terminal domain of focal adhesion kinase: a potential role for protein kinase A.
Focal adhesion kinase (pp125(FAK)) is a protein tyrosine kinase that is localized to focal adhesions in many cell types and which undergoes tyrosine phosphorylation after integrin binding to extracellular matrix. In some cells the C-terminal non-catalytic domain of pp125(FAK) is expressed as a separate protein referred to as FRNK (FAK-related, non-kinase). We have previously shown that overexpression of FRNK inhibits tyrosine phosphorylation of pp125(FAK) and its substrates as well as inhibiting cell spreading on fibronectin. In this report we identify Ser148 and Ser151 as residues in FRNK that are phosphorylated after tyrosine phosphorylation of pp125(FAK) and in response to integrin binding to fibronectin. Tyrosine phosphorylation of pp125(FAK) appears to be an early event after integrin occupancy, and serine phosphorylation of FRNK occurs significantly later. Treatment of fibroblasts with a series of protein kinase A inhibitors delayed serine phosphorylation of FRNK as well as cell spreading on fibronectin and tyrosine phosphorylation of pp125(FAK). However, these PKA inhibitors are unlikely to delay cell spreading simply by preventing serine phosphorylation of FRNK, as overexpression of FRNK containing mutations of Ser148 and Ser151 either singly or jointly to either alanine or glutamate residues did not significantly alter the ability of FRNK to act as an inhibitor of pp125(FAK).
Richardson
A
A
Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA.
Shannon
J D
JD
Adams
R B
RB
Schaller
M D
MD
Parsons
J
J
D000818:Animals; D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D051416:Focal Adhesion Protein-Tyrosine Kinases; D016023:Integrins / Q000502:physiology*; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D015815:Cell Adhesion Molecules; D016023:Integrins; D010446:Peptide Fragments; D010748:Phosphopeptides; D011994:Recombinant Proteins; D010768:Phosphoserine; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases