?url_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Adc&rft.relation=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F22819%2F&rft.title=Structural+modeling+of+regulation+in+%CE%B1-actinin%2FF-actin+interactions&rft.creator=Travers%2C+Timothy+Ni%C3%B1o+S.&rft.description=The+%CE%B1-actinins+(ACTNs)+are+a+highly+conserved+family+of+actin-crosslinking+proteins+that+are+critical+to+various+fundamental+biological+processes+in+eukaryotes%2C+ranging+from+cell+motility+and+surface+remodeling+to+muscle+contraction.+Binding+of+ACTNs+to+actin+filaments+is+regulated+by+several+mechanisms%3A+epidermal+growth+factor+(EGF)-induced+tyrosine+phosphorylation%2C+binding+of+calcium%2C+limited+proteolysis+by+calpain+enzymes%2C+and+binding+of+phosphoinositide+moieties.+The+molecular+mechanisms+by+which+these+external+cues+drive+the+regulation+of+ACTN+function+are+still+not+understood%2C+however%2C+largely+because+there+is+currently+no+high-resolution+experimental+structure+that+brings+together+the+multiple+domains+that+comprise+ACTNs.+An+understanding+of+these+molecular+mechanisms+should+provide+us+with+insights+into+how+the+cell+is+able+to+modulate+actin+cytoskeletal+remodeling+and+give+rise+to+complex+cellular+phenomena.%0D%0A%0D%0AIn+this+thesis%2C+we+investigate+how+these+external+cues+regulate+the+actin-binding+function+of+human+ACTN4%2C+a+non-muscle+isoform+that+is+essential+to+cell+motility+and+has+been+implicated+in+cancer+invasion+and+metastasis.+First%2C+we+develop+and+validate+an+atomic+model+of+the+multi-domain+assembly+that+makes+up+the+full+ACTN4+homodimer%2C+with+a+novel+ternary+complex+between+CH2%2C+neck%2C+and+CaM2+comprising+the+core+of+this+assembly.+Next%2C+we+show+that+a+novel+tandem+phosphorylation+mechanism+in+the+disordered+N-terminal+region+of+ACTN4%2C+where+phosphorylation+of+the+functional+Y31+requires+prior+phosphorylation+at+Y4%2C+is+responsible+for+the+regulation+of+ACTN4+function+in+the+presence+of+EGF.+This+tandem+mechanism+can+work+in+conjunction+with+m-calpain+cleavage+of+the+N-terminal+to+generate+varied+actin-binding+responses+at+the+front+and+rear+ends+of+the+cell+during+motility.+Using+our+full+structural+model%2C+we+also+show+that%3A+(i)+Y265+phosphorylation+eases+ABD+opening%3A+(ii)+binding+of+calcium+may+break+the+CaM2%2Fneck+complex%3B+(iii)+CaM2+protects+the+neck+region+from+m-calpain+cleavage%3B+and+(iv)+binding+of+phosphoinositides+to+CH2+allows+ACTN4+to+crosslink+actin+filaments+at+the+inner+membrane.+Finally%2C+we+bring+together+these+structural+insights+to+develop+a+preliminary+network-level+model+that+can+serve+as+a+computational+tool+for+predicting+the+actin-binding+response+of+ACTN4+in+the+presence+of+multiple+external+cues.&rft.date=2014-08-22&rft.type=University+of+Pittsburgh+ETD&rft.type=PeerReviewed&rft.format=application%2Fpdf&rft.language=en&rft.identifier=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F22819%2F1%2FTraversTimothy_ETD2014.pdf&rft.identifier=++Travers%2C+Timothy+Ni%C3%B1o+S.++(2014)+Structural+modeling+of+regulation+in+%CE%B1-actinin%2FF-actin+interactions.++Doctoral+Dissertation%2C+University+of+Pittsburgh.++++(Unpublished)++