IntroductionThrombopoietin (Tpo) is critical for the maintenance of hematopoietic stem and progenitor cells and is also the primary regulator of megakaryocyte development. 1,2 The binding of Tpo to its receptor, c-Mpl, causes associated Janus kinase 2 (Jak2) activation, which in turn phosphorylates (activates) several downstream effectors including signal transducers and activators of transcription (STAT) 3 and 5, mitogen-activated protein kinase (MAPK), phosphotidylinositol-3-kinase (PI3-K), and protein kinase C (PKC). [3][4][5][6][7] Activation of these pathways promotes proliferation and survival in c-Mplexpressing cell lines and hematopoietic progenitor cells, in addition to megakaryocyte lineage differentiation and maturation. [8][9][10][11] It is critical that Tpo signal transduction is stringently controlled to prevent uncontrolled proliferation. Suppressors of cytokine signaling (SOCS) proteins, phosphatases, and negative regulators such FAK, Lnk, and Lyn have all been shown to down-modulate Tpo-induced signaling. [12][13][14][15] However, the most effective method of regulating Tpo signaling is by controlling expression of c-Mpl on the plasma membrane. Tpo-mediated c-Mpl endocytosis, recycling, and degradation are rapid mechanisms to control signaling longevity and represent a mechanism that regulates Tpo signaling without new protein expression.The principal mechanism of receptor-mediated endocytosis in eukaryotic cells is the clathrin-coated vesicle. 16 Soluble clathrin molecules self-assemble and are recruited to the plasma membrane, where they form lattice structures and interact with transmembrane receptors via adaptor proteins (APs), such as AP2, to form clathrin-coated pits. 17,18 These pits then further invaginate before finally budding from the membrane to form clathrin-coated vesicles. AP2 is a heterotetramer composed of ␣2, 2, 2, and 2 subunits. The ␣2 and 2 subunits localize AP2 to the membrane, recruit endocytic accessory proteins, and bind clathrin heavy chain. [19][20][21] Transmembrane proteins are associated with the AP2-clathrin complex via the 2 domain, which binds directly to cytoplasmic YXX⌽ (where X ϭ any amino acid and ⌽ ϭ bulky hydrophobic residue) and [DE]XXX-L[IL] motifs. 22,23 To ensure that AP2 specifically associates with membrane-bound proteins, phosphorylation of 156 Thr in the 2 subunit results in a conformational change in AP2, dramatically increasing the affinity of 2 for YXX⌽ motifs. 24,25 156 Thr is phosphorylated by adaptor-associated kinase 1 (AAK1), 26 the activity of which is maximized by its association with clathrin, 27,28 ensuring that AP2-cargo protein interactions are initiated only at the plasma membrane. In addition to being an endocytic signal, YXX⌽ motifs located between 6 to 9 amino acids from the transmembrane domain mediate targeting of cargo protein to the lysosome and lysosome-like organelles via interactions with AP3. [29][30][31][32] c Methods Chemicals and reagentsPharmacologic inhibitors JakI, LY294002, SU6656, and U0126 where all purchased f...
The current observational study provides descriptive data on 270 pressure injuries (PrIs) among 142 racially/ethnically diverse nursing home (NH) residents over 16 weeks. Weekly assessments were conducted with the Bates-Jensen Wound Assessment Tool. NH data were obtained from public government websites. NH, resident, and PrI characteristics across race/ethnicity groups were compared using analysis of variance and chi-square. Participants were 62% female and 89% functionally dependent. More Black and Asian individuals had peripheral vascular disease. More Black individuals had persistent trunk and Stage 4 PrIs. Black and Hispanic individuals had normal skin color surrounding PrIs. More Asian individuals had PrIs surrounded by purple/red discolored skin. More Black individuals' heel PrIs were unstageable, necrotic, and showed no granulation. Black and Hispanic individuals exhibited more deep tissue injury. No NH or prevention differences existed. Health disparities found validate administrative data results. Differences in PrI characteristics should be further examined among diverse NH residents. [ Journal of Gerontological Nursing, 47 (3), 37–46.]
Thrombopoietin (TPO), acting through its receptor Mpl, promotes survival and proliferation of hematopoietic progenitor cells and also drives megakaryocyte differentiation. The pro-proliferation and survival signals activated by TPO must therefore be tightly regulated to prevent uncontrolled cell growth. Several mechanisms to down-regulate hematopoietic growth factor signaling have been identified, including increased expression of suppressors of cytokine signaling (SOCS) proteins, activation of protein phosphatases, and endocytosis and degradation of activated growth factor receptors. In this work we determined the mechanisms that control TPO-stimulated Mpl internalization and defined the processes leading to Mpl degradation using IL-3-dependent BaF3 cells engineered to express wild type (WT) and mutant forms of human Mpl. Stimulation of BaF-Mpl cells with TPO lead to rapid endocytosis of Mpl which was blocked by pre-treatment with the clathrin-mediated endocytosis inhibitor monodansylcadaverine, indicating that Mpl internalization is clathrin-mediated. Additionally, we found that inhibition of Janus kinase 2 (Jak2) and Src-family kinases greatly reduced Mpl internalization. Sites of clathrin assembly on the plasma membrane contain the adaptor protein complex AP2, which associates with transmembrane proteins enabling targeted endocytosis. Association of AP2 and transmembrane proteins relies on an interaction between AP2μ2 subunit and internalization motifs YXXΦ (where X=any amino acid and Φ=bulky hydrophobic) expressed by target proteins. Mpl contains two YXXΦ motifs, at cytoplasmic Y8 (Y8RRL) and Y78 (Y78RRL). BaF-Mpl cells transfected with siRNA targeted to AP2 displayed greatly reduced TPO-mediated internalization of Mpl, demonstrating that AP2-Mpl association is critical for Mpl endocytosis. Next, we introduced Y to F point mutations at Mpl cytoplasmic Y8 and Y78 individually, and in combination (Y8+78F). Internalization was greatly reduced in Mpl Y78F and Mpl Y8+78F after TPO stimulation, whereas Mpl Y8F internalization was only slightly attenuated. Furthermore, we found that Mpl Y78F and Y8+78F exhibited increased proliferation and increased strength and duration of activated Jak2 and AKT in response to TPO, compared to Mpl WT and Y8F. In addition to mediating endocytosis, cytoplasmic YXXΦ motifs located between 6 and 9 residues from the transmembrane domain have been shown to mediate lysosomal targeting of proteins following endocytosis. Consequently, we studied the role of Mpl Y8RRL motif in Mpl lysosome targeting. BaF-Mpl WT and Y8F cells were pretreated with cyclohexamide and stimulated with TPO for up to 2 hours. Mpl Y8F displayed greatly reduced Mpl degradation in response to TPO compared to WT. Furthermore, Mpl Y8F recycled back to the plasma membrane following TPO starvation quicker than WT Mpl, suggesting that the Mpl Y8F remains in endosomes, rather than being targeted to lysosomes. Our data shows that Mpl cytoplasmic YRRL motifs are responsible for both TPO-mediated internalization via interactions with AP2 and lysosomal targeting following endocytosis. These findings significantly advance our understanding of normal Mpl function. Further study of internalization motifs, which are present in receptors for other hematopoietic growth factors including; erythropoietin, granulocyte colony stimulating factor, leukemia inhibitory factor and interleukin, may highlight the importance of these sequences in mediating hematopoiesis and potentially aid identification of novel targets in hematological disease.
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