EGF-like peptide-enhanced cell movement in Dictyostelium is mediated by protein kinases and the activity of several cytoskeletal proteins

• Published in: Cellular signalling 2012-09, Vol.24 (9), p.1770-1780
• Main Authors: Huber, Robert J., O'Day, Danton H.
• Format: Article
• Language: English
• Subjects: Cell motility; Cell Movement - drug effects; Cyra; Cytoskeletal Proteins - metabolism; Cytoskeleton; Dictyostelium; Dictyostelium - cytology; Dictyostelium - drug effects; Dictyostelium - enzymology; Dictyostelium - metabolism; Dictyostelium discoideum; EGF-like peptide; Epidermal Growth Factor - chemistry; Peptides - chemistry; Peptides - pharmacology; Phosphorylation; Protein Kinases - metabolism; Signal Transduction - drug effects; Structure-Activity Relationship; Vinculin B
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/j.cellsig.2012.05.004

DdEGFL1, a synthetic epidermal growth factor-like (EGFL) peptide based on the first EGFL repeat of the extracellular matrix, cysteine-rich, calmodulin-binding protein CyrA, has previously been shown to sustain the threonine phosphorylation of a 210kDa protein during the starvation of Dictyostelium cells. Immunoprecipitation coupled with a LC/MS/MS analysis identified the 210kDa protein as vinculin B (VinB). VinB shares sequence similarity with mammalian vinculin, a protein that links the actin cytoskeleton to the plasma membrane. Both threonine phosphorylated VinB (P-VinB) and VinB-GFP localized to the cytoplasm and cytoskeleton of Dictyostelium amoebae. VinB-GFP was also shown to be threonine phosphorylated and co-immunoprecipitated with established vinculin-binding cytoskeletal proteins (e.g. myosin II heavy chain, actin, alpha-actinin, talin). P-VinB and VinB-GFP were detected in DdEGFL1 pull-down assays, which also identified a 135kDa phosphothreonine protein and two phosphotyrosine proteins (35 and 32kDa) as potential components of the DdEGFL1 signaling pathway. DdEGFL1-enhanced cell movement required the cytoskeletal proteins talin B and paxillin B and tyrosine kinase activity mediated by PKA signaling, however VinB threonine phosphorylation was shown to be independent of PI3K/PLA2 signaling and PI3K and PKA kinase activity. Finally, VinB-GFP over-expression suppressed DdEGFL1-enhanced random cell movement, but not folic acid-mediated chemotaxis. Together, this study provides the first evidence for VinB function plus new insight into the signaling pathway(s) mediating EGFL repeat/peptide-enhanced cell movement in Dictyostelium. This information is integrated into an emerging model that summarizes existing knowledge. ► VinB threonine phosphorylation mediates DdEGFL1-enhanced cell movement. ► VinB localizes to the cytoplasm and cytoskeleton of Dictyostelium amoebae. ► VinB interacts with DdEGFL1 and known vinculin-binding cytoskeletal proteins. ► DdEGFL1 signaling requires cytoskeletal proteins talin and paxillin. ► DdEGFL1 signaling requires PKA tyrosine kinase activity.