title: Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics.
abstract: Activation of cyclic nucleotide dependent signaling pathways leads to relaxation of smooth muscle, alterations in the cytoskeleton of cultured cells, and increases in the phosphorylation of HSP20. To determine the effects of phosphorylated HSP20 on the actin cytoskeleton, phosphopeptide analogs of HSP20 were synthesized. These peptides contained 1) the amino acid sequence surrounding the phosphorylation site of HSP20, 2) a phosphoserine, and 3) a protein transduction domain. Treatment of Swiss 3T3 cells with phosphopeptide analogs of HSP20 led to loss of actin stress fibers and focal adhesion complexes as demonstrated by immunocytochemistry, interference reflection microscopy, and biochemical quantitation of globular-actin. Treatment with phosphopeptide analogs of HSP20 also led to dephosphorylation of the actin depolymerizing protein cofilin. Pull-down assays demonstrated that 14-3-3 proteins associated with phosphopeptide analogs of HSP20 (but not peptide analogs in which the serine was not phosphorylated). The binding of 14-3-3 protein to phosphopeptide analogs of HSP20 prevented the association of cofilin with 14-3-3. These data suggest that HSP20 may modulate actin cytoskeletal dynamics by competing with the actin depolymerizing protein cofilin for binding to the scaffolding protein 14-3-3. Interestingly, the entire protein was not needed for this effect, suggesting that the association is modulated by phosphopeptide motifs of HSP20. These data also suggest the possibility that cyclic nucleotide dependent relaxation of smooth muscle may be mediated by a thin filament (actin) regulatory process. Finally, these data suggest that protein transduction can be used as a tool to elucidate the specific function of peptide motifs of proteins.
authors.affiliation: The Biodesign Institute at Arizona State University, Tempe, Arizona, USA.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Dreiza
authors.forename: Colleen M
authors.initials: CM
authors.lastname: Brophy
authors.forename: Padmini
authors.initials: P
authors.lastname: Komalavilas
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Furnish
authors.forename: Lokesh
authors.initials: L
authors.lastname: Joshi
authors.forename: Manuel A
authors.initials: MA
authors.lastname: Pallero
authors.forename: Joanne E
authors.initials: JE
authors.lastname: Murphy-Ullrich
authors.forename: Moritz
authors.initials: M
authors.lastname: von Rechenberg
authors.forename: Yew-seng J
authors.initials: YS
authors.lastname: Ho
authors.forename: Bonnie
authors.initials: B
authors.lastname: Richardson
authors.forename: Nafei
authors.initials: N
authors.lastname: Xu
authors.forename: Yuejun
authors.initials: Y
authors.lastname: Zhen
authors.forename: John M
authors.initials: JM
authors.lastname: Peltier
authors.forename: Alyssa
authors.initials: A
authors.lastname: Panitch
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D016475:3T3 Cells / Q000737:chemistry / Q000378:metabolism; D051339:Actin Depolymerizing Factors; D000185:Actinin / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D022001:Focal Adhesions / Q000378:metabolism; D050886:HSP20 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D051419:Paxillin; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016596:Vinculin / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D051339:Actin Depolymerizing Factors; D000199:Actins; D003598:Cytoskeletal Proteins; D050886:HSP20 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008840:Microfilament Proteins; D051419:Paxillin; D010455:Peptides; D010748:Phosphopeptides; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D000185:Actinin; D016596:Vinculin; D010768:Phosphoserine