title: Properties and catalytic activities of MICAL1, the flavoenzyme involved in cytoskeleton dynamics, and modulation by its CH, LIM and C-terminal domains.
abstract: MICAL1 is a cytoplasmic 119 kDa protein participating in cytoskeleton dynamics through the NADPH-dependent oxidase and F-actin depolymerizing activities of its N-terminal flavoprotein domain, which is followed by calponin homology (CH), LIM domains and a C-terminal region with Pro-, Glu-rich and coiled-coil motifs. MICAL1 and truncated forms lacking the C-terminal, LIM and/or CH regions have been produced and characterized. The CH, LIM and C-terminal regions cause an increase of Km,NADPH exhibited by the NADPH oxidase activity of the flavoprotein domain, paralleling changes in the overall protein charge. The C-terminus also determines a ∼ 10-fold decrease of kcat, revealing its role in establishing an inactive/active conformational equilibrium, which is at the heart of the regulation of MICAL1 in cells. F-actin lowers Km,NADPH (10-50 μM) and increases kcat (10-25 s(-1)) to similar values for all MICAL forms. The apparent Km,actin of MICAL1 is ∼ 10-fold higher than that of the other forms (3-5 μM), reflecting the fact that F-actin binds to the flavoprotein domain in the MICAL's active conformation and stabilizes it. Analyses of the reaction in the presence of F-actin indicate that actin depolymerization is mediated by H2O2 produced by the NADPH oxidase reaction, rather than due to direct hydroxylation of actin methionine residues.
authors: Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133, Milano, Italy.
authors: Teresa
authors: T
authors: Vitali
authors: Filarete Foundation, Via Ortles 22, 20139, Milano, Italy.
authors: Elisa
authors: E
authors: Maffioli
authors: Filarete Foundation, Via Ortles 22, 20139, Milano, Italy; Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università degli Studi di Milano, Via Celoria 10, 20133, Milano, Italy.
authors: Gabriella
authors: G
authors: Tedeschi
authors: Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133, Milano, Italy. Electronic address: maria.vanoni@unimi.it.
authors: Maria A
authors: MA
authors: Vanoni
mesh_terms: D000199:Actins / Q000737:chemistry; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry*; D000818:Animals; D055162:Biocatalysis; D003598:Cytoskeletal Proteins / Q000737:chemistry*; D003599:Cytoskeleton / Q000737:chemistry*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D060588:LIM Domain Proteins / Q000737:chemistry*; D008840:Microfilament Proteins; D006899:Mixed Function Oxygenases; D008958:Models, Molecular; D019255:NADPH Oxidases / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry; D014783:Viscosity; D000096985:Calponins
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; D006861:Hydrogen Peroxide; D060588:LIM Domain Proteins; D008840:Microfilament Proteins; D006899:Mixed Function Oxygenases; D019255:NADPH Oxidases; D011994:Recombinant Proteins; C455272:MICAL1 protein, human; D000096985:Calponins
keywords: Cytoskeleton; F-actin depolymerization; FAD-containing monooxygenase/oxidase; Flavoprotein; MICAL1; Semaphorin signaling