Phosphorylation of Ser 21 in Fyn regulates its kinase activity, focal adhesion targeting, and is required for cell migration

Abstract

The tyrosine kinase Fyn is a member of the Src family kinases which are important in many integrin-mediated cellular processes including cell adhesion and migration. Fyn has multiple phosphorylation sites which can affect its kinase activity. Among these phosphorylation sites, the serine 21 (S21) residue of Fyn is a protein kinase A (PKA) recognition site within an RxxS motif of the amino terminal SH4 domain of Fyn. In addition, S21 is critical for Fyn kinase-linked cellular signaling. Mutation of S21A blocks PKA phosphorylation of Fyn and alters its tyrosine kinase activity. Expression of Fyn S21A in cells lacking Src family kinases (SYF cell) led to decreased tyrosine phosphorylation of focal adhesion kinase resulting in reduced focal adhesion targeting, which slowed lamellipodia dynamics and thus cell migration. These changes in cell motility were reflected by the fact that cells expressing Fyn S21A were severely deficient in their ability to assemble and disassemble focal adhesions. Taken together, our findings indicate that phosphorylation of S21 within the pPKA recognition site (RxxS motif) of Fyn regulates its tyrosine kinase activity and controls focal adhesion targeting, and that this residue of Fyn is critical for transduction of signals arising from cell-extracellular matrix interactions.

Low-Intensity Pulsed Ultrasound Enhances Angiogenesis and Ameliorates Left Ventricular Dysfunction in a Mouse Model of Acute Myocardial Infarction

“…[23][24][25] Although caveolin-1 has no extracellular component, caveolin-1 may play an important role in sensing mechanical stress or the distortion of the extracellular membranes through interaction with β1-integrin. 19,20,26,27 In the present study, we aimed to demonstrate whether the LIPUS ameliorates post-MI LV remodeling and if so, to elucidate the underlying molecular mechanisms involved in the beneficial effects of the LIPUS.…”

Anchoring of Protein Kinase A by ERM (Ezrin-Radixin-Moesin) Proteins Is Required for Proper Netrin Signaling through DCC (Deleted in Colorectal Cancer)

“…In this regard, it is intriguing to note that a considerable number of proteins implicated in netrin/DCC signaling are known to be regulated, directly or indirectly, by PKA (9,10,32,72). These include but are not limited to the non-receptor tyrosine kinases FAK, Src, and Fyn (42,(73)(74)(75)(76)(77)(78), the adaptor protein Nck (79,80), the Rho family GTPases Rac and Cdc42 (27,45,(81)(82)(83) and their effector PAK (p21-activated kinase) (42,84), and as shown here, the Mena/VASP proteins (35,41,46). Also of particular interest is the well established cross-talk between cAMP/PKA and Ca 2ϩ signaling in cell migration (33) and the observed intricate interplay between localized cAMP (and perhaps PKA) and Ca 2ϩ signaling in growth cone turning responses (18,85), although additional work is needed to determine the relative contributions of PKA versus other cAMP effectors (principally, the Epacs (exchange proteins activated by cAMP) (21,85)) in this context.…”

Role of Focal Adhesions in Lamellipodia Dynamics

“…Furthermore, experiments in p130CAS-null MEFs show the lamellipodia protrusion and ruffle retraction velocity decreases compared with wt p130CAS MEFs [18]. The Fyn-null MEFs also show that the lamellipodia protrusion and ruffle retraction velocity decreases compared to the wt Fyn MEFs [13]. These SFK deficient MEFs commonly show less formation of FAs and reduced lamellipodia protrusion and retraction velocity, but membrane persistence times are prolonged compared to wt MEFs.…”

“…This computer-assisted stroboscopic analysis of cell dynamics (SACED) is used for analyzing lamellipodia and ruffle formation after the cell adheres to the ECM [4,17,18] (Figure 1) In the comparative studies on defective FA formation in MEFs, these MEFs commonly show longer lamellipodia persistence time than wild type (wt) MEFs. The SACED analysis of persistence time (sec; mean ± SD) of Crk-null MEFs is 40.30s ± 1.7 and wt Crk is 25.82s ± 0.66, SYF MEF is 90.81s ± 27.79 and wt Fyn (recovered Fyn cDNA in SYF MEF) [13] is 65.25s ± 38.46, and p130CAS-null MEF is 67.2 s ± 10.1083 and wt p130CAS is 36.5455s ± 6.3303 [13,15,18]. Furthermore, experiments in p130CAS-null MEFs show the lamellipodia protrusion and ruffle retraction velocity decreases compared with wt p130CAS MEFs [18].…”

“…Deficiency of p130CAS causes severe defects in cell spreading [9,10]. In SYF cells (deficient for Src, Yes, and Fyn), MEFs show a decreased formation of FAs, which results in severe developmental defects, lethality, and delayed cell migration [11][12][13]. Additionally, Crknull mice die during a late stage of embryonic development [14] and present with a defect in cell-spreading in vitro [15].…”