Mitophagy is an important type of selective autophagy for specific elimination of damaged mitochondria. PTEN-induced putative kinase protein 1 (PINK1)-catalyzed phosphorylation of ubiquitin (Ub) plays a critical role in the onset of PINK1-Parkin-mediated mitophagy. Phosphatase and tensin homolog (PTEN)-long (PTEN-L) is a newly identified isoform of PTEN, with addition of 173 amino acids to its N-terminus. Here we report that PTEN-L is a novel negative regulator of mitophagy via its protein phosphatase activity against phosphorylated ubiquitin. We found that PTEN-L localizes at the outer mitochondrial membrane (OMM) and overexpression of PTEN-L inhibits, whereas deletion of PTEN-L promotes, mitophagy induced by various mitochondria-damaging agents. Mechanistically, PTEN-L is capable of effectively preventing Parkin mitochondrial translocation, reducing Parkin phosphorylation, maintaining its closed inactive conformation, and inhibiting its E3 ligase activity. More importantly, PTEN-L reduces the level of phosphorylated ubiquitin (pSer65-Ub) in vivo, and in vitro phosphatase assay confirms that PTEN-L dephosphorylates pSer65-Ub via its protein phosphatase activity, independently of its lipid phosphatase function. Taken together, our findings demonstrate a novel function of PTEN-L as a protein phosphatase for ubiquitin, which counteracts PINK1-mediated ubiquitin phosphorylation leading to blockage of the feedforward mechanisms in mitophagy induction and eventual suppression of mitophagy. Thus, understanding this novel function of PTEN-L provides a key missing piece in the molecular puzzle controlling mitophagy, a critical process in many important human diseases including neurodegenerative disorders such as Parkinson's disease.
Bile canaliculi expand and contract in response to the amount of secreted bile, and resistance from the surrounding actin bundles. Further expansion due to bile duct blockade leads to the formation of inward blebs, which carry away excess bile to prevent bile build up in the canaliculi.
Chemotaxis in shallow gradients of chemoattractants is accomplished by preferential maintenance of protrusions oriented towards the chemoattractant; however, the mechanism of preferential maintenance is not known. Here, we test the hypothesis that kinectindependent endoplasmic reticulum (ER) transport supports focal complex maturation to preferentially maintain correctly oriented protrusions. We knocked down kinectin expression in MDA-MB-231 cells using small interfering RNA and observed that kinectin contributes to the directional bias, but not the speed, of cell migration. Kymograph analysis revealed that the extension of protrusions oriented towards the chemoattractant was not affected by kinectin knockdown, but that their maintenance was. Immunofluorescence staining and live-cell imaging demonstrated that kinectin transports ER preferentially to protrusions oriented towards the chemoattractant. ER then promotes the maturation of focal complexes into focal adhesions to maintain these protrusions for chemotaxis. Our results show that kinectin-dependent ER distribution can be localized by chemoattractants and provide a mechanism for biased protrusion choices during chemotaxis in shallow gradients of chemoattractants.
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