Two distinct phosphorylation events govern the function of muscle FHOD3.
Posttranslational modifications such as phosphorylation are universally acknowledged regulators of protein function. Recently we characterised a striated muscle-specific isoform of the formin FHOD3 that displays distinct subcellular targeting and protein half-life compared to its non-muscle counterpart and which is dependent on phosphorylation by CK2 (formerly casein kinase 2). We now show that the two isoforms of FHOD3 are already expressed in the vertebrate embryonic heart. Analysis of CK2 alpha knockout mice showed that phosphorylation by CK2 is also required for proper targeting of muscle FHOD3 to the myofibrils in embryonic cardiomyocytes in situ. The localisation of muscle FHOD3 in the sarcomere varies depending on the maturation state, being either broader or restricted to the Z-disc proper in the adult heart. Following myofibril disassembly, such as that in dedifferentiating adult rat cardiomyocytes in culture, the expression of non-muscle FHOD3 is up-regulated, which is reversed once the myofibrils are reassembled. The shift in expression levels of different isoforms is accompanied by an increased co-localisation with p62, which is involved in autophagy, and affects the half-life of FHOD3. Phosphorylation of three amino acids in the C-terminus of FHOD3 by ROCK1 is sufficient for activation, which results in increased actin filament synthesis in cardiomyocytes and also a broader localisation pattern of FHOD3 in the myofibrils. ROCK1 can directly phosphorylate FHOD3, and FHOD3 seems to be the downstream mediator of the exaggerated actin filament formation phenotype that is induced in cardiomyocytes upon the overexpression of constitutively active ROCK1. We conclude that the expression of the muscle FHOD3 isoform is characteristic of the healthy mature heart and that two distinct phosphorylation events are crucial to regulate the activity of this isoform in thin filament assembly and maintenance.
Muscle Cell Biology Section, The Randall Division of Cell and Molecular Biophysics and The Cardiovascular Division, BHF Research Excellence Centre, King's College London, New Hunt's House, Guy's Campus, London, SE1 1UL, UK.
Thomas
T
Iskratsch
Susan
S
Reijntjes
Joseph
J
Dwyer
Paul
P
Toselli
Irene R
IR
Dégano
Isabel
I
Dominguez
Elisabeth
E
Ehler
D000818:Animals; D030161:Avian Proteins / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D047390:Casein Kinase II / Q000235:genetics; D002478:Cells, Cultured; D002642:Chick Embryo / Q000196:embryology / Q000378:metabolism; D002645:Chickens; D002522:Chlorocebus aethiops; D000080865:Formins; D018507:Gene Expression Regulation, Developmental; D006367:HeLa Cells; D006321:Heart / Q000196:embryology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism / Q000648:ultrastructure; D020033:Protein Isoforms / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D051381:Rats; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism
D030161:Avian Proteins; C485994:FHOD3 protein, human; C574082:Fhod3 protein, mouse; C579478:Fhod3 protein, rat; D000080865:Formins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D020033:Protein Isoforms; D047390:Casein Kinase II; C515851:ROCK1 protein, human; C578418:ROCK1 protein, rat; D054460:rho-Associated Kinases