Publications by authors named "Einat Levy-Apter"

4 Publications

  • Page 1 of 1

Production of Osteoclasts for Studying Protein Tyrosine Phosphatase Signaling.

Methods Mol Biol 2016 ;1447:283-300

Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, 76100, Israel.

Osteoclasts, specialized cells that degrade bone, are key components of the cellular system that regulates and maintains bone homeostasis. Aberrant function of osteoclasts can lead to pathological loss or gain of bone mass, such as in osteopetrosis, osteoporosis, and several types of cancer that metastasize to bone. Phosphorylation of osteoclast proteins on tyrosine residues is critical for formation of osteoclasts and for their proper function and responses to physiological signals. Here we describe preparation and growth of osteoclasts from bone marrow of mice, use of viral vectors to downregulate expression of endogenous proteins and to express exogenous proteins in osteoclasts, and analysis of signaling processes triggered by M-CSF, estrogen, and physical contact with matrix in these cells.
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http://dx.doi.org/10.1007/978-1-4939-3746-2_16DOI Listing
January 2018

Adaptor protein GRB2 promotes Src tyrosine kinase activation and podosomal organization by protein-tyrosine phosphatase ϵ in osteoclasts.

J Biol Chem 2014 Dec 7;289(52):36048-58. Epub 2014 Nov 7.

From the Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel,

The non-receptor isoform of protein-tyrosine phosphatase ϵ (cyt-PTPe) supports adhesion of bone-resorbing osteoclasts by activating Src downstream of integrins. Loss of cyt-PTPe reduces Src activity in osteoclasts, reduces resorption of mineralized matrix both in vivo and in cell culture, and induces mild osteopetrosis in young female PTPe KO mice. Activation of Src by cyt-PTPe is dependent upon this phosphatase undergoing phosphorylation at its C-terminal Tyr-638 by partially active Src. To understand how cyt-PTPe activates Src, we screened 73 Src homology 2 (SH2) domains for binding to Tyr(P)-638 of cyt-PTPe. The SH2 domain of GRB2 bound Tyr(P)-638 of cyt-PTPe most prominently, whereas the Src SH2 domain did not bind at all, suggesting that GRB2 may link PTPe with downstream molecules. Further studies indicated that GRB2 is required for activation of Src by cyt-PTPe in osteoclast-like cells (OCLs) in culture. Overexpression of GRB2 in OCLs increased activating phosphorylation of Src at Tyr-416 and of cyt-PTPe at Tyr-638; opposite results were obtained when GRB2 expression was reduced by shRNA or by gene inactivation. Phosphorylation of cyt-PTPe at Tyr-683 and its association with GRB2 are integrin-driven processes in OCLs, and cyt-PTPe undergoes autodephosphorylation at Tyr-683, thus limiting Src activation by integrins. Reduced GRB2 expression also reduced the ability of bone marrow precursors to differentiate into OCLs and reduced the fraction of OCLs in which podosomal adhesion structures assume organization typical of active, resorbing cells. We conclude that GRB2 physically links cyt-PTPe with Src and enables cyt-PTPe to activate Src downstream of activated integrins in OCLs.
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http://dx.doi.org/10.1074/jbc.M114.603548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4276870PMC
December 2014

Protein tyrosine phosphatases ε and α perform nonredundant roles in osteoclasts.

Mol Biol Cell 2014 Jun 2;25(11):1808-18. Epub 2014 Apr 2.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

Female mice lacking protein tyrosine phosphatase ε (PTP ε) are mildly osteopetrotic. Osteoclasts from these mice resorb bone matrix poorly, and the structure, stability, and cellular organization of their podosomal adhesion structures are abnormal. Here we compare the role of PTP ε with that of the closely related PTP α in osteoclasts. We show that bone mass and bone production and resorption, as well as production, structure, function, and podosome organization of osteoclasts, are unchanged in mice lacking PTP α. The varying effects of either PTP on podosome organization in osteoclasts are caused by their distinct N-termini. Osteoclasts express the receptor-type PTP α (RPTPa), which is absent from podosomes, and the nonreceptor form of PTP ε (cyt-PTPe), which is present in these structures. The presence of the unique 12 N-terminal residues of cyt-PTPe is essential for podosome regulation; attaching this sequence to the catalytic domains of PTP α enables them to function in osteoclasts. Serine 2 within this sequence regulates cyt-PTPe activity and its effects on podosomes. We conclude that PTPs α and ε play distinct roles in osteoclasts and that the N-terminus of cyt-PTPe, in particular serine 2, is critical for its function in these cells.
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http://dx.doi.org/10.1091/mbc.E14-03-0788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038506PMC
June 2014

Protein tyrosine phosphatase epsilon affects body weight by downregulating leptin signaling in a phosphorylation-dependent manner.

Cell Metab 2011 May;13(5):562-72

Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel.

Molecular-level understanding of body weight control is essential for combating obesity. We show that female mice lacking tyrosine phosphatase epsilon (RPTPe) are protected from weight gain induced by high-fat food, ovariectomy, or old age and exhibit increased whole-body energy expenditure and decreased adiposity. RPTPe-deficient mice, in particular males, exhibit improved glucose homeostasis. Female nonobese RPTPe-deficient mice are leptin hypersensitive and exhibit reduced circulating leptin concentrations, suggesting that RPTPe inhibits hypothalamic leptin signaling in vivo. Leptin hypersensitivity persists in aged, ovariectomized, and high-fat-fed RPTPe-deficient mice, indicating that RPTPe helps establish obesity-associated leptin resistance. RPTPe associates with and dephosphorylates JAK2, thereby downregulating leptin receptor signaling. Leptin stimulation induces phosphorylation of hypothalamic RPTPe at its C-terminal Y695, which drives RPTPe to downregulate JAK2. RPTPe is therefore an inhibitor of hypothalamic leptin signaling in vivo, and provides controlled negative-feedback regulation of this pathway following its activation.
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http://dx.doi.org/10.1016/j.cmet.2011.02.017DOI Listing
May 2011
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