title: EGF-like peptide-enhanced cell movement in Dictyostelium is mediated by protein kinases and the activity of several cytoskeletal proteins.
abstract: 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.
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON, Canada M5S 3G5.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Huber
authors.forename: Danton H
authors.initials: DH
authors.lastname: O'Day
mesh_terms: D002465:Cell Movement / Q000187:drug effects*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004023:Dictyostelium / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013329:Structure-Activity Relationship
chemical_list: D003598:Cytoskeletal Proteins; D010455:Peptides; D004815:Epidermal Growth Factor; D011494:Protein Kinases