Molecule Interacting With CasL Research Articles

Abstract B017: MICAL2 promotes pancreatic cancer growth and metastasis independent of MRTF-A signaling

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in large part due to its propensity for early metastasis. We used ChIP-seq to identify differentially tagged super-enhancer associated genes in PDAC and identified MICAL2 as a gene of interest. MICAL (molecule interacting with CasL) proteins are flavin monooyxgenases that induce actin depolymerization. MICAL2 canonically modulates nuclear actin to regulate SRF/MRTF-A (myocardin related transcription factor) mediated transcription to drive EMT (epithelial-mesenchymal transition) and fibrosis. In this study, we sought to determine the impact of MICAL2 on PDAC progression and its dependence on SRF/MRTF-A signaling. Methods: Studies were performed in human and murine PDAC models. MICAL2 and MRTF-A were knocked down with shRNA and gene expression quantitated with qPCR. In vitro cell viability was assessed by MTT colorimetric assay. Cell migration was tested with in vitro wound scratch assays. Cell cycle analysis was done with flow cytometry-based DNA content measurement. Subcutaneous, orthotopic, and intrasplenic injections of PDAC cells were performed to assess in vivo growth and metastasis. Protein expression was measured with Western blotting. Student’s t-test was used to confirm statistical significance of experimental results. Results: In human PDAC cell lines, MICAL2 knockdown decreased cell viability, reduced cell migration and wound healing, and induced cell cycle arrest at G0/G1. MICAL2 inhibition was associated with decreased p-AKT, c-Myc, and increased p27 by Western blot. Orthotopic and subcutaneous injections of KPC46 shMICAL2 cells were performed in syngeneic mice; orthotopic injections of shMICAL2 cells led to decreased tumor growth compared to control (0.26 gm vs. 1 gm, p = 0.008), not observed with shMRTF-A (1.032 gm vs. 1 gm, p = 0.95). Subcutaneous injections of MICAL2 knockdown cells led to no tumor growth; injections of MRTF-A knockdown cells again led to comparable tumor growth as control (3.02 gm vs. 1.72 gm, p = 0.24). GSEA (Gene Set Enrichment Analysis) of RNAseq data demonstrated a correlation between MICAL2 knockdown and downregulation of Hallmark EMT pathways. Intrasplenic injection studies in syngeneic mice revealed grossly decreased liver metastases after intrasplenic injection of shMRTF-A cells, with total abrogation of metastases after injection of shMICAL2 cells. Conclusions: MICAL2 promotes pancreatic cancer cell growth and metastasis. While MICAL2’s effect on metastasis appears largely dependent on SRF/MRTF-A, tumor growth appears to be MRTF-A independent, suggesting putative novel functions of MICAL2. Given its potentially targetable enzymatic domain and important role in promoting cell growth in vitro and tumor progression and metastasis in vivo, MICAL2 holds promise as a novel tractable therapeutic target in pancreatic cancer. Citation Format: Sohini Khan, Shweta Sharma, Divya Sood, Bharti Garg, Dawn Jaquish, Evangeline Mose, Edgar Esparza, Herve Tiriac, Andrew M. Lowy. MICAL2 promotes pancreatic cancer growth and metastasis independent of MRTF-A signaling [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B017.

Flavin monooxygenases regulate Caenorhabditis elegans axon guidance and growth cone protrusion with UNC-6/Netrin signaling and Rac GTPases.

The guidance cue UNC-6/Netrin regulates both attractive and repulsive axon guidance. Our previous work showed that in C. elegans, the attractive UNC-6/Netrin receptor UNC-40/DCC stimulates growth cone protrusion, and that the repulsive receptor, an UNC-5:UNC-40 heterodimer, inhibits growth cone protrusion. We have also shown that inhibition of growth cone protrusion downstream of the UNC-5:UNC-40 repulsive receptor involves Rac GTPases, the Rac GTP exchange factor UNC-73/Trio, and the cytoskeletal regulator UNC-33/CRMP, which mediates Semaphorin-induced growth cone collapse in other systems. The multidomain flavoprotein monooxygenase (FMO) MICAL (Molecule Interacting with CasL) also mediates growth cone collapse in response to Semaphorin by directly oxidizing F-actin, resulting in depolymerization. The C. elegans genome does not encode a multidomain MICAL-like molecule, but does encode five flavin monooxygenases (FMO-1, -2, -3, -4, and 5) and another molecule, EHBP-1, similar to the non-FMO portion of MICAL. Here we show that FMO-1, FMO-4, FMO-5, and EHBP-1 may play a role in UNC-6/Netrin directed repulsive guidance mediated through UNC-40 and UNC-5 receptors. Mutations in fmo-1, fmo-4, fmo-5, and ehbp-1 showed VD/DD axon guidance and branching defects, and variably enhanced unc-40 and unc-5 VD/DD axon guidance defects. Developing growth cones in vivo of fmo-1, fmo-4, fmo-5, and ehbp-1 mutants displayed excessive filopodial protrusion, and transgenic expression of FMO-5 inhibited growth cone protrusion. Mutations suppressed growth cone inhibition caused by activated UNC-40 and UNC-5 signaling, and activated Rac GTPase CED-10 and MIG-2, suggesting that these molecules are required downstream of UNC-6/Netrin receptors and Rac GTPases. From these studies we conclude that FMO-1, FMO-4, FMO-5, and EHBP-1 represent new players downstream of UNC-6/Netrin receptors and Rac GTPases that inhibit growth cone filopodial protrusion in repulsive axon guidance.

MICAL—methionine sulfoxide reductase couple: a new target for the development of neuroprotective strategies

The flavin adenine dinucleotide-containing domain of the protein MICAL (molecule interacting with CasL) is a monooxygenase. This protein plays an important role in the regulation of axonal guidance. However, the mechanism of this process has been unknown until recently. Only two years ago, F-actin was found to be the physiological substrate. The oxidation of methionine residues of this substrate is catalyzed by MICAL, resulting in the depolymerization of actin. A year ago it was shown that methionine sulfoxide reductase (MSR) catalyzes the reverse reaction of reduction of oxidized actin methionines. Therefore, this couple of proteins is currently a target for manipulation of the ability of neurons to regenerate their axons. The present study deals with different approaches to the regulation of the activity of MICAL and MSR and the design of inhibitors of the former enzyme.

The intracellular redox protein MICAL-1 regulates the development of hippocampal mossy fibre connections.

Mical is a reduction-oxidation (redox) enzyme that functions as an unusual F-actin disassembly factor during Drosophila development. Although three Molecule interacting with CasL (MICAL) proteins exist in vertebrate species, their mechanism of action remains poorly defined and their role in vivo unknown. Here, we report that vertebrate MICAL-1 regulates the targeting of secretory vesicles containing immunoglobulin superfamily cell adhesion molecules (IgCAMs) to the neuronal growth cone membrane through its ability to control the actin cytoskeleton using redox chemistry, thereby maintaining appropriate IgCAM cell surface levels. This precise regulation of IgCAMs by MICAL-1 is essential for the lamina-specific targeting of mossy fibre axons onto CA3 pyramidal neurons in the developing mouse hippocampus in vivo. These findings reveal the first in vivo role for a vertebrate MICAL protein, expand the repertoire of cellular functions controlled through MICAL-mediated effects on the cytoskeleton, and provide insights into the poorly characterized mechanisms underlying neuronal protein cell surface expression and lamina-specific axonal targeting.

Structural Insights into Modulation of Mical by its Calponin Homology (CH) Domain

MICAL (Molecule Interacting with CasL) is a 1048 amino acid protein consisting of a monooxygenase domain (FD) with redox activity, a Calponin homology domain (CH), a LIM domain, a proline-rich region, and a C-term region containing coiled-coil ERM α-like domain. In axon guidance, MICAL is a key molecule that links the extracellular signal from semaphorins -a class of repulsive guidance cues- to the reorganization of the cytoskeleton. Proper axon guidance, the process by which growing axons respond to extracellular cues that guide them towards their appropriate targets, is vital in neural development processes such as neuronal cell-migration, axonal branching, path finding, and fasiculation/defasiculation. Our laboratory has previously determined the crystal structure of MICALs FD domain (MICALFD) and showed that it uses NADPH as the reductant. Studies showed that MICALFD and MICALFD-CH can bind and oxidize Met44 on actin filaments, thereby affecting their polymerization dynamics. However, modulation of these MICAL activities by its non-redox domains is poorly understood. To structurally characterize the modulation by the CH domain, we determined the crystal structure of MICALFD-CH to 3.0-A resolution. The structure reveals that the CH domain does not interact with the active site in the FD domain. Furthermore, the FD and CH domains are flexible with respect to each other; MICALFD-CH crystallized in two different crystal forms, and no electron density was observed for the 18-residue linker between the two domains. In actin-binding proteins with tandem CH domains, the flexibility of the domains with respect to each other is important for binding F-actin. Similarly, the flexibility of the two domains in MICALFD-CH may be important in optimizing the binding to F-actin such that Met44 is more accessible to the active site.

MICAL, the Flavoenzyme Participating in Cytoskeleton Dynamics

MICAL (from the Molecule Interacting with CasL) indicates a family of recently discovered cytosolic, multidomain proteins, which uniquely couple an N-terminal FAD-containing monooxygenase-like domain to typical calponine homology, LIM and coiled-coil protein-interaction modules. Genetic and cell biology approaches have demonstrated an essential role of the catalytic activity of the monooxygenase-like domain in transducing the signal initiated by semaphorins interaction with their plexin receptors, which results in local actin cytoskeleton disassembly as part of fundamental processes that include differentiation, migration and cell-cell contacts in neuronal and non-neuronal cell types. This review focuses on the structure-function relations of the MICAL monooxygenase-like domain as they are emerging from the available in vitro studies on mouse, human and Drosophila MICAL forms that demonstrated a NADPH-dependent actin depolymerizing activity of MICAL. With Drosophila MICAL forms, actin depolymerization was demonstrated to be associated to conversion of Met44 to methionine sulfone through a postulated hydroxylating reaction. Arguments supporting the concept that MICAL effect on F-actin may be reversible will be discussed.

Extracellular inhibitors, repellents, and semaphorin/plexin/MICAL-mediated actin filament disassembly.

Multiple extracellular signals have been identified that regulate actin dynamics within motile cells, but how these instructive cues present on the cell surface exert their precise effects on the internal actin cytoskeleton is still poorly understood. One particularly interesting class of these cues is a group of extracellular proteins that negatively alter the movement of cells and their processes. Over the years, these types of events have been described using a variety of terms and herein we provide an overview of inhibitory/repulsive cellular phenomena and highlight the largest known protein family of repulsive extracellular cues, the Semaphorins. Specifically, the Semaphorins (Semas) utilize Plexin cell-surface receptors to dramatically collapse the actin cytoskeleton and we summarize what is known of the direct molecular and biochemical mechanisms of Sema-triggered actin filament (F-actin) disassembly. We also discuss new observations from our lab that reveal that the multidomain oxidoreductase (Redox) enzyme Molecule Interacting with CasL (MICAL), an important mediator of Sema/Plexin repulsion, is a novel F-actin disassembly factor. Our results indicate that MICAL triggers Sema/Plexin-mediated reorganization of the F-actin cytoskeleton and suggest a role for specific Redox signaling events in regulating actin dynamics.

MICAL-like1 mediates epidermal growth factor receptor endocytosis.

Small GTPase Rabs are required for membrane protein sorting/delivery to precise membrane domains. Rab13 regulates epithelial tight junction assembly and polarized membrane transport. Here we report that Molecule Interacting with CasL (MICAL)-like1 (MICAL-L1) interacts with GTP-Rab13 and shares a similar domain organization with MICAL. MICAL-L1 has a calponin homology (CH), LIM, proline rich and coiled-coil domains. It is associated with late endosomes. Time-lapse video microscopy shows that green fluorescent protein-Rab7 and mcherry-MICAL-L1 are present within vesicles that move rapidly in the cytoplasm. Depletion of MICAL-L1 by short hairpin RNA does not alter the distribution of a late endosome/lysosome-associated protein but affects the trafficking of epidermal growth factor receptor (EGFR). Overexpression of MICAL-L1 leads to the accumulation of EGFR in the late endosomal compartment. In contrast, knocking down MICAL-L1 results in the distribution of internalized EGFR in vesicles spread throughout the cytoplasm and promotes its degradation. Our data suggest that the N-terminal CH domain associates with the C-terminal Rab13 binding domain (RBD) of MICAL-L1. The binding of Rab13 to RBD disrupts the CH/RBD interaction, and may induce a conformational change in MICAL-L1, promoting its activation. Our results provide novel insights into the MICAL-L1/Rab protein complex that can regulate EGFR trafficking at late endocytic pathways.

Characterizing the novel protein p33MONOX

The novel protein p33MONOX (p33Monooxygenase) was over-expressed in neuroblastoma cells demonstrating its inhibitory effect on the phosphorylation of the App (amyloid precursor protein) and Bcl2 (B-cell lymphoma 2) proteins but mediating higher activation of Mapk1/3 (mitogen-activated protein kinase 1/3). We employed a variety of cell biology techniques to show the localization of p33MONOX to the cytoplasm of pyramidal neurons in the mouse brain hippocampus. We also carried out a yeast-two-hybrid screening plus co-immunoprecipitation and bio-informatics to determine COBRA1 (cofactor of BRCA1 (breast cancer type 1)), NOL12 (nucleolar protein 12), and PRNP (prion protein) as p33MONOX-interacting proteins. Bio-computational analyses revealed a flavine-containing monooxygenase (FMO)-1 motif, thus linking p33MONOX to a group of previously characterized proteins, the MICALs (molecule interacting with CasL). Concluding, p33MONOX might regulate pre- and post-transcriptional control of dynamic processes related to growth cone guidance.

Mechanism for the Selective Interaction of C-terminal Eps15 Homology Domain Proteins with Specific Asn-Pro-Phe-containing Partners

Epidermal growth factor receptor tyrosine kinase substrate 15 (Eps15) homology (EH)-domain proteins can be divided into two classes: those with an N-terminal EH-domain(s), and the C-terminal Eps15 homology domain-containing proteins (EHDs). Whereas many N-terminal EH-domain proteins regulate internalization events, the best characterized C-terminal EHD, EHD1, regulates endocytic recycling. Because EH-domains interact with the tripeptide Asn-Pro-Phe (NPF), it is of critical importance to elucidate the molecular mechanisms that allow EHD1 and its paralogs to interact selectively with a subset of the hundreds of NPF-containing proteins expressed in mammalian cells. Here, we capitalize on our findings that C-terminal EH-domains possess highly positively charged interaction surfaces and that many NPF-containing proteins that interact with C-terminal (but not N-terminal) EH-domains are followed by acidic residues. Using the recently identified EHD1 interaction partner molecule interacting with CasL (MICAL)-Like 1 (MICAL-L1) as a model, we have demonstrated that only the first of its two NPF motifs is required for EHD1 binding. Because only this first NPF is followed by acidic residues, we have utilized glutathione S-transferase pulldowns, two-hybrid analysis, and NMR to demonstrate that the flanking acidic residues "fine tune" the binding affinity to EHD1. Indeed, our NMR solution structure of the EHD1 EH-domain in complex with the MICAL-L1 NPFEEEEED peptide indicates that the first two flanking Glu residues lie in a position favorable to form salt bridges with Lys residues within the EH-domain. Our data provide a novel explanation for the selective interaction of C-terminal EH-domains with specific NPF-containing proteins and allow for the prediction of new interaction partners with C-terminal EHDs.

Regulation of epithelial cell adhesion and repulsion: role of endocytic recycling

A proper balance between cell adhesion and repulsion is essential for cellular morphogenesis during epithelial-mesenchymal transition and mesenchymal-epithelial transition. A number of ligand-receptor pairs including hepatocyte growth factor/scatter factor-Met and semaphorin-plexin are known to control this balance through the complex intracellular signaling pathways. Cell adhesion to other cells and extracellular matrix (ECM) is mediated by cell adhesion molecules (CAMs) and ECM receptors, respectively, which are associated with cytoskeleton through a variety of plaque proteins strengthening and/or weakening adhesion activities. Cell repulsion requires the downregulation of cell adhesion and the extensive changes in cytoskeletal dynamics. The endocytic recycling of CAMs and ECM receptors has recently emerged as an important mechanism to control the balance between cell adhesion and repulsion. Molecule interacting with CasL (MICAL) family proteins are originally identified as a plaque protein associated with ECM receptors integrins and implicated in semaphorin-plexin dependent repulsive axon guidance. We have recently shown that MICAL family protein JRAB/MICAL-L2 functions as an effector protein for Rab family small G protein Rab13 and regulates the endocytic recycling of tight junctional CAM occludin and controls the adhesion and repulsion of epithelial cells.

High-resolution structure of the catalytic region of MICAL (molecule interacting with CasL), a multidomain flavoenzyme-signaling molecule

Semaphorins are extracellular cell guidance cues that govern cytoskeletal dynamics during neuronal and vascular development. MICAL (molecule interacting with CasL) is a multidomain cytosolic protein with a putative flavoprotein monooxygenase (MO) region required for semaphorin-plexin repulsive axon guidance. Here, we report the 1.45-A resolution crystal structure of the FAD-containing MO domain of mouse MICAL-1 (residues 1-489). The topology most closely resembles that of the NADPH-dependent flavoenzyme p-hydroxybenzoate hydroxylase (PHBH). Comparison of structures before and after reaction with NADPH reveals that, as in PHBH, the flavin ring can switch between two discrete positions. In contrast with other MOs, this conformational switch is coupled with the opening of a channel to the active site, suggestive of a protein substrate. In support of this hypothesis, distinctive structural features highlight putative protein-binding sites in suitable proximity to the active site entrance. The unusual juxtaposition of this N-terminal MO (hydroxylase) activity with the characteristics of a multiprotein-binding scaffold exhibited by the C-terminal portion of the MICALs represents a unique combination of functionality to mediate signaling.

MICAL, a Novel CasL Interacting Molecule, Associates with Vimentin

CasL/HEF1 belongs to the p130(Cas) family. It is tyrosine-phosphorylated following beta(1) integrin and/or T cell receptor stimulation and is thus considered to be important for immunological reactions. CasL has several structural motifs such as an SH3 domain and a substrate domain and interacts with many molecules through these motifs. To obtain more insights on the CasL-mediated signal transduction, we sought proteins that interact with the CasL SH3 domain by far Western screening, and we identified a novel human molecule, MICAL (a Molecule Interacting with CasL). MICAL is a protein of 118 kDa and is expressed in the thymus, lung, spleen, kidney, testis, and hematopoietic cells. MICAL has a calponin homology domain, a LIM domain, a putative leucine zipper motif, and a proline-rich PPKPP sequence. MICAL associates with CasL through this PPKPP sequence. MICAL is a cytoplasmic protein and colocalizes with CasL at the perinuclear area. Through the COOH-terminal region, MICAL also associates with vimentin that is a major component of intermediate filaments. Immunostaining revealed that MICAL localizes along with vimentin intermediate filaments. These results suggest that MICAL may be a cytoskeletal regulator that connects CasL to intermediate filaments.