title: Enzymatic cleavage of myoferlin releases a dual C2-domain module linked to ERK signalling.
abstract: Myoferlin and dysferlin are closely related members of the ferlin family of Ca2+-regulated vesicle fusion proteins. Dysferlin is proposed to play a role in Ca2+-triggered vesicle fusion during membrane repair. Myoferlin regulates endocytosis, recycling of growth factor receptors and adhesion proteins, and is linked to the metastatic potential of cancer cells. Our previous studies establish that dysferlin is cleaved by calpains during membrane injury, with the cleavage motif encoded by alternately-spliced exon 40a. Herein we describe the cleavage of myoferlin, yielding a membrane-associated dual C2 domain 'mini-myoferlin'. Myoferlin bears two enzymatic cleavage sites: a canonical cleavage site encoded by exon 38 within the C2DEdomain; and a second cleavage site in the linker adjacent to C2DE, encoded by alternately-spliced exon 38a, homologous to dysferlin exon 40a. Both myoferlin cleavage sites, when introduced into dysferlin, can functionally substitute for exon 40a to confer Ca2+-triggered calpain cleavage in response to membrane injury. However, enzymatic cleavage of myoferlin is complex, showing both constitutive or Ca2+-enhanced cleavage in different cell lines, that is not solely dependent on calpains-1 or -2. The functional impact of myoferlin cleavage was explored through signalling protein phospho-protein arrays revealing specific activation of ERK1/2 by ectopic expression of cleavable myoferlin, but not an uncleavable isoform. In summary, we molecularly define two enzymatic cleavage sites within myoferlin and demonstrate 'mini-myoferlin' can be detected in human breast cancer tumour samples and cell lines. These data further illustrate that enzymatic cleavage of ferlins is an evolutionarily preserved mechanism to release functionally specialized mini-modules.
authors.affiliation: Institute for Neuroscience and Muscle Research, Children's Hospital at Westmead, Sydney, NSW 2145, Australia; Discipline of Child and Adolescent Health, Faculty of Medicine, University of Sydney, Sydney, Australia.
authors.forename: Ann-Katrin
authors.initials: AK
authors.lastname: Piper
authors.affiliation: Institute for Neuroscience and Muscle Research, Children's Hospital at Westmead, Sydney, NSW 2145, Australia.
authors.forename: Samuel E
authors.initials: SE
authors.lastname: Ross
authors.affiliation: EMBL Australia Node in Single Molecule Science, School of Medical Science, University of New South Wales, Sydney, NSW, Australia.
authors.forename: Gregory M
authors.initials: GM
authors.lastname: Redpath
authors.affiliation: Institute for Neuroscience and Muscle Research, Children's Hospital at Westmead, Sydney, NSW 2145, Australia.
authors.forename: Frances A
authors.initials: FA
authors.lastname: Lemckert
authors.affiliation: Institute for Neuroscience and Muscle Research, Children's Hospital at Westmead, Sydney, NSW 2145, Australia; Discipline of Child and Adolescent Health, Faculty of Medicine, University of Sydney, Sydney, Australia.
authors.forename: Natalie
authors.initials: N
authors.lastname: Woolger
authors.affiliation: Institute for Neuroscience and Muscle Research, Children's Hospital at Westmead, Sydney, NSW 2145, Australia.
authors.forename: Adam
authors.initials: A
authors.lastname: Bournazos
authors.affiliation: Department of Pathology and Molecular Medicine, Queen's University, Division of Cancer Biology and Genetics, Queen's Cancer Research Institute, Kingston, ON K7L 3N6, Canada.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Greer
authors.affiliation: Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA; Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA.
authors.forename: Roger B
authors.initials: RB
authors.lastname: Sutton
authors.affiliation: Institute for Neuroscience and Muscle Research, Children's Hospital at Westmead, Sydney, NSW 2145, Australia; Discipline of Child and Adolescent Health, Faculty of Medicine, University of Sydney, Sydney, Australia. Electronic address: sandra.cooper@sydney.edu.au.
authors.forename: Sandra T
authors.initials: ST
authors.lastname: Cooper
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D045744:Cell Line, Tumor; D000073939:Dysferlin / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020935:MAP Kinase Signaling System; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D000072417:Protein Domains; D059748:Proteolysis; D014162:Transfection
chemical_list: D002135:Calcium-Binding Proteins; C114839:DYSF protein, human; D000073939:Dysferlin; C415584:MYOF protein, human; D008565:Membrane Proteins; D009124:Muscle Proteins; D002154:Calpain
keywords: Breast cancer; Calpain; Cell signalling; Extracellular-signal-regulated kinase (ERK); Myoferlin; Proteolysis