Molecular and functional characterization of MICAL proteins

Abstract

Since their original identification in 2002, MICAL proteins have been implicated in various physiological and pathological processes including axon guidance, tight junction formation, spinal cord injury and cancer. MICALs mediate cell signaling via their unusual N-terminal monooxygenase (MO) domain and various C-terminal protein-protein interacting modules, including a Calponin Homology (CH) domain, a LIM domain and coiled-coil motifs (CC). The constitutively active MO domain (i.e. C-terminally truncated MICAL-1) catalyzes the synthesis of hydrogen peroxide (H2O2) in presence of NADPH. In mammalian cells, the MO domain induces cell contraction but this effect does not depend on H2O2. This result supports the idea that the effects of MICAL-1 on cell morphology are mediated by direct redox modification of potential substrates such as CRMP and F-actin. The MICAL C-terminal domains have been shown to regulate the enzymatic activity of the MO domain. We further reported two novel molecular properties of MICAL-1. First, MICAL-1 can form dimers or oligomers most likely through the CH domain. This self-association may regulate MICAL-1 activity or function in the association with binding partners. Second, MICAL-1 is a phospho-protein both in cell lines and in brain tissue. Serine 777 was identified as one of the phosphorylation sites in mouse MICAL-1. This finding indicates that MICALs may be regulated by protein kinases. The multidomain protein structure of MICAL suggests a role as a scaffold protein with the potential to recruit multiple distinct signaling and structural proteins into signaling complexes that mediate various cellular processes. To further characterize the signaling pathways in which MICALs participate, we used different approaches to purify MICAL-1-containing signaling complexes from mammalian cells followed by mass spectrometry to identify potential MICAL-1 binding proteins. The various putative MICAL-1 interactors identified implicate previously unknown roles for MICAL-1 in cellular processes such as cytoskeletal remodeling, tight junction formation, apoptosis, transcriptional and translational regulation. Several novel interactors, including NDR1, DOCK7 and staufen have been confirmed by protein-interaction studies of individual proteins. Among the candidate interactors identified, NDR kinases were further studied in more detail. NDR kinases (NDR1/2) did not influence enzymatic activity of MICAL-1, however, MICAL-1 negatively regulated NDR1/2 phosphorylation and therefore activity. This new mechanism of negative NDR kinase regulation was dependent on the competitive binding of MICAL-1 to the C-terminal part NDR1/2 which is also targeted by MST1 kinase. MST1 normally activates NDR1/2 by phosphorylating Threonine 444/442 (in NDR1/2). MICAL-1 prevents MST1 binding and thereby negatively regulates the pro-apoptotic effects mediated by the MST1-NDR pathway. These findings unveil a previously unknown biological role for MICAL-1 in apoptosis and define a novel negative regulatory mechanism of MST-NDR signaling. Finally, we observed overlapping expression patterns for MICAL-1 and NDR1 in the developing brain and provided data to suggest that MICAL-1, NDR1 and activated plexinA1 exist in a tripartite protein complex. Overall, our data contribute to a further understanding of MICAL proteins and their signaling roles and form an important basis for future studies into the role and actions of these unique intracellular proteins.