Inactivated SRC was maintained at the ERC. Knockdown analysis in HeLa cells demonstrated that EHD1 was required for SRC transport and activation and acted as a molecular 'pinchase' on MICALL1 tubules to release SRC from the ERC in response to EGF. Further analysis revealed that MICALL1 regulated cell spreading and was required for migration of human foreskin fibroblasts. The results indicated that MICALL1 regulates transport of active SRC to focal adhesions, thereby controlling turnover of focal adhesions. MICALL1 was also required for PDGF-induced focal adhesion turnover.
Likewise, MICALL1 colocalized with SRC in human foreskin fibroblasts and was required for SRC recruitment to circular dorsal ruffles (CDRs) following platelet-derived growth factor (PDGF see 173430) stimulation. MICALL1 interacted with SRC and was required for SRC activation and transport from perinuclear region to the plasma membrane upon epidermal growth factor (EGF 131530) stimulation. (2014) showed that MICALL1 colocalized with SRC ( 190090) along tubular membranes that radiated from the ERC in HeLa cells under steady-state conditions. Using immunofluorescence analysis, Reinecke et al. The author concluded that MICALL1 is involved in sorting/targeting EGFR to the recycling pathway. In contrast, Micall1 knockdown resulted in distribution of internalized Egfr in vesicles spread throughout the cytoplasm and promoted Egfr degradation. Overexpression of human MICALL1 led to accumulation of Egfr in late endosomal compartments. Knockdown of endogenous Micall1 in MDCK cells did not alter distribution of tight junction proteins, but it affected trafficking of epidermal growth factor receptor (EGFR 131550). MICALL1 was associated with late and recycling endosomes in transfected MDCK cells, with rapid movement in cytoplasm. Using yeast 2-hybrid screening, pull-down assays, and immunofluorescence analysis in transfected Madin-Darby canine kidney (MDCK) cells, Zahraoui (2014) showed that human MICALL1 interacted and colocalized with RAB13 ( 602672), but that MICALL1 did not function as a GTPase-activating protein for RAB13. Further analysis demonstrated that MICALL and SYND2 were capable of generating tubules from phosphatidic acid-containing membranes. A lipid overlay assay revealed that MICALL1 and SYND2 bound to phosphatidic acid in membranes of REs for localization and for RE tubule biogenesis. Knockdown analysis showed that interaction between MICALL1 and SYND2 was required for their localization to tubular REs, whereas EHD1 stabilized interaction between MICALL1 and SYND2 on recycling tubules. SYND2 colocalized with MICALL1 and EHD1 at tubular recycling endosomes (REs), with SYND2 and MICALL1 displaying similar dynamics of association with tubular membranes. (2013) demonstrated that the SH3 domain of SYND2 (SDC2 142460) interacted directly with 2 of the 14 proline-rich domains (PRDs) of MICALL1. Using immunoprecipitation analysis in HeLa cells, Giridharan et al.
MICALL1 recruited and linked EHD1 and RAB8A ( 165040) on membrane tubules, thereby regulating endocytic recycling from the endocytic recycling compartment (ERC) to the plasma membrane. In contrast, MICALL1 was required for association of EHD1 with tubular membranes. However, association of MICALL1 with tubular membranes was independent of EHD1 and EHD3, as the C-terminal coiled-coli domain of MICALL1 alone was essential and sufficient for tubular localization. MICALL1 and EHD1 colocalized at tubular membranes in HeLa cells and were dynamically recruited to tubular membranes with similar kinetics. The interaction required the first NPF motif of MICALL1. Using pull-down, yeast 2-hybrid, and coimmunoprecipitation analyses, Sharma et al.
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