Abstract
The canonical protein tyrosine phosphatase PTP1B is an important regulator of diverse cellular signaling networks. PTP1B has long been thought to exert its influence solely from its perch on the endoplasmic reticulum (ER); however, an additional subpopulation of PTP1B has recently been detected in mitochondria extracted from rat brain tissue. Here, we show that PTP1B’s mitochondrial localization is general (observed across diverse mammalian cell lines) and sensitively dependent on the transmembrane domain length, C-terminal charge and hydropathy of its short (≤35 amino acid) tail anchor. Our electron microscopy of specific DAB precipitation revealed that PTP1B localizes via its tail anchor to the outer mitochondrial membrane (OMM), with fluorescence lifetime imaging microscopy establishing that this OMM pool contributes to the previously reported cytoplasmic interaction of PTP1B with endocytosed epidermal growth factor receptor. We additionally examined the mechanism of PTP1B’s insertion into the ER membrane through heterologous expression of PTP1B’s tail anchor in wild-type yeast and yeast mutants of major conserved ER insertion pathways: In none of these yeast strains was ER targeting significantly impeded, providing in vivo support for the hypothesis of spontaneous membrane insertion (as previously demonstrated in vitro). Further functional elucidation of the newly recognized mitochondrial pool of PTP1B will likely be important for understanding its complex roles in cellular responses to external stimuli, cell proliferation and diseased states.
Highlights
The founding member of its family, protein tyrosine phosphatase 1B (PTP1B) [1,2] is an important regulator of phosphotyrosine signaling in mammalian cells through its dephosphorylation of a range of substrates [4], including the receptors for insulin, leptin and epidermal growth factor (EGF) and their downstream substrates; the tyrosine kinases JAK2 and c-Src; and the tyrosine phosphatase SHP2
Recent evidence from Western blots and electron microscopy has demonstrated the localization of PTP1B in mitochondria that were extracted from rat brain tissue [32,33]
The challenges that we encountered in discriminating mitochondria-specific subpopulations of PTP1B from its more general endoplasmic reticulum (ER) staining in particular cell lines offers a possible explanation for the fact that its mitochondrial localization was not discovered in earlier fluorescence-based studies [10,12]
Summary
The founding member of its family, protein tyrosine phosphatase 1B (PTP1B) [1,2] (the protein product of the gene PTPN1 [3]) is an important regulator of phosphotyrosine signaling in mammalian cells through its dephosphorylation of a range of substrates [4], including the receptors for insulin, leptin and epidermal growth factor (EGF) and their downstream substrates; the tyrosine kinases JAK2 and c-Src; and the tyrosine phosphatase SHP2. PTP1B is expressed as two separate splice variants [8], the first identified in rat brain tissue [9] with the second later identified in human placenta [5] These variants differ only in their terminal amino acids, with the first variant ending in VCFH and the second in FLFNSNT. PTP1B’s insertion into the ER has been shown in vitro to proceed in the absence of membrane proteins [13] and in vivo to at least partially involve the chaperones Hsp40/ Hsc70 [14], the latter in agreement with other tail anchor proteins [15]. In addition to the two different splice variants, further diversity of PTP1B, which might affect its subcellular targeting, is generated through several post-translational modifications that are known to activate or inhibit it [4], including phosphorylation (on multiple serines and tyrosines), oxidation, sumoylation and proteolysis (calpain cleavage)
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