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The inositol phosphatase MTMR4 is a novel target of the ubiquitin ligase Nedd4.

Plant PJ, Correa J, Goldenberg N, Bain J, Batt J - Biochem. J. (2009)

Bottom Line: The inositol phosphatase, MTMR4 (myotubularin-related protein 4), was identified as a novel interactor of the ubiquitin ligase Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4). hMTMR4 (human MTMR4) and Nedd4 co-immunoprecipitated and co-localized to late endosomes.The PY (Pro-Tyr) motif of hMTMR4 binds to WW (Trp-Trp) domains of hNedd4.MTMR4 expression was decreased in atrophying muscle, whereas Nedd4 expression was increased and hMTMR4 was ubiquitinated by hNedd4, suggesting that this novel interaction may underlie the biological process of muscle breakdown.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Clinical Sciences Division, Room 7344, Medical Science Building, University of Toronto, Toronto, 1 Kings College Circle, Ontario, Canada.

ABSTRACT
The inositol phosphatase, MTMR4 (myotubularin-related protein 4), was identified as a novel interactor of the ubiquitin ligase Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4). hMTMR4 (human MTMR4) and Nedd4 co-immunoprecipitated and co-localized to late endosomes. The PY (Pro-Tyr) motif of hMTMR4 binds to WW (Trp-Trp) domains of hNedd4. MTMR4 expression was decreased in atrophying muscle, whereas Nedd4 expression was increased and hMTMR4 was ubiquitinated by hNedd4, suggesting that this novel interaction may underlie the biological process of muscle breakdown.

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MTMR4 is a PY-motif-containing protein expressed in muscle that interacts with Nedd4(A) Schematic diagram of human MTMR4, based on [33]. Protein domains predicted with SMART (http://smart.embl-heidelberg.de/) and PFSCAN (http://hits.isb-sib.ch/cgi-bin/PFSCAN) include GRAM (glucosyltransferase, Rab-like GTPase activator of myotubularins) domain, RID (Rac-induced recruitment domain), PTP/DSP domain, SID (SET-interacting domain), PY motif (XPPXY), CC (coiled-coil) domain and FYVE domain. (B) Western blot of hMTMR4, Nedd4 and tubulin from transfected (MTM tx) or untransfected (untx) HEK-293 lysate immunoprecipitated with anti-MTMR4 antibodies (MTM I.P.). Equal amounts of whole lysate were run as a control. (C) Lysates (lysate) and immunoprecipitates using anti-Nedd4 antibodies (Nedd4 I.P.) or anti-(p70 S6 kinase) antibodies (p70 S6 I.P.) as negative control, of HeLa cells transfected with HA–hMTMR4 wild-type (MTM WT), HA–hMTMR4 PY mutant (MTM PY), pcDNA 3.1 vector alone (vec) or untransfected (untx). Immunoblotting (IB) was performed with anti-HA (monoclonal) antibodies followed by anti-Nedd4 (polyclonal) antibodies and blots were stripped and re-probed with anti-tubulin antibodies as control. Molecular masses are indicated in kDa.
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Figure 1: MTMR4 is a PY-motif-containing protein expressed in muscle that interacts with Nedd4(A) Schematic diagram of human MTMR4, based on [33]. Protein domains predicted with SMART (http://smart.embl-heidelberg.de/) and PFSCAN (http://hits.isb-sib.ch/cgi-bin/PFSCAN) include GRAM (glucosyltransferase, Rab-like GTPase activator of myotubularins) domain, RID (Rac-induced recruitment domain), PTP/DSP domain, SID (SET-interacting domain), PY motif (XPPXY), CC (coiled-coil) domain and FYVE domain. (B) Western blot of hMTMR4, Nedd4 and tubulin from transfected (MTM tx) or untransfected (untx) HEK-293 lysate immunoprecipitated with anti-MTMR4 antibodies (MTM I.P.). Equal amounts of whole lysate were run as a control. (C) Lysates (lysate) and immunoprecipitates using anti-Nedd4 antibodies (Nedd4 I.P.) or anti-(p70 S6 kinase) antibodies (p70 S6 I.P.) as negative control, of HeLa cells transfected with HA–hMTMR4 wild-type (MTM WT), HA–hMTMR4 PY mutant (MTM PY), pcDNA 3.1 vector alone (vec) or untransfected (untx). Immunoblotting (IB) was performed with anti-HA (monoclonal) antibodies followed by anti-Nedd4 (polyclonal) antibodies and blots were stripped and re-probed with anti-tubulin antibodies as control. Molecular masses are indicated in kDa.

Mentions: As Nedd4 overexpression was correlated with the progression of atrophic processes in muscle cells, we sought potential substrates in muscle that may be targeted by Nedd4 to elicit its effects. To this end, we took a bioinformatics approach to elucidate PY-motif-containing proteins that were expressed in skeletal muscle. Nedd4 consists of an N-terminal C2 domain, three or four WW domains and the ubiquitin ligase HECT domain and, in many cases, engages in interactions with its substrates via a WW domain–PY motif interaction [4–9]. Searching a SIDNET (Signalling Identification Network) MGC (Mammalian Gene Collection) clone database (http://www.sickkids.ca/Research/SIDNET/SIDNET.html) for PY-motif-containing proteins that were expressed in skeletal muscle limited potential substrates of Nedd4 to several proteins, including MTMR4, which belongs to a family of lipid tyrosine phosphatases with specificity towards PtdIns3P and PtdIns(3,5)P2 [14,15]. MTMR4 possesses several protein–protein and protein–lipid interaction domains (Figure 1A) including the FYVE domain, shown to mediate its localization to endosomes through its interaction with PtdIns3P and PtdIns(3,5)P2 [14] and is the only family member possessing a PY motif. The MTMR4 PY-motif (VPPLY) at the C-terminus (positions 1004–1008) is conserved across mammalian species (results not shown), adhering to the canonical L/XPPXY consensus sequence for WW domain type I binding. To verify that Nedd4 and MTMR4 can interact, cDNA for hMTMR4 was transfected into HEK-293 cells, and lysate from these cells was subjected to immunoprecipitation with anti-MTMR4 antibodies. Immunoprecipitates were separated by SDS/PAGE and probed with anti-MTMR4 and anti-Nedd4 antibodies to determine whether the two proteins interacted, with anti-tubulin antibodies used as a control. Immunoprecipitated transfected hMTMR4 interacted with endogenous Nedd4, but no band was detected in untransfected lysate (Figure 1B). Similarly, cell lysate transfected with HA-tagged hMTMR4 wild-type and PY-motif mutant was immunoprecipitated with anti-Nedd4 antibodies, and subjected to Western blotting with anti-Nedd4, anti-HA and anti-tubulin antibodies. Endogenous Nedd4 was able to immunoprecipitate transfected wild-type hMTMR4, but not the MTMR4 PY mutant, suggesting that the endogenous association of the two proteins is likely to be mediated through the WW domain–PY motif interaction (Figure 1C).


The inositol phosphatase MTMR4 is a novel target of the ubiquitin ligase Nedd4.

Plant PJ, Correa J, Goldenberg N, Bain J, Batt J - Biochem. J. (2009)

MTMR4 is a PY-motif-containing protein expressed in muscle that interacts with Nedd4(A) Schematic diagram of human MTMR4, based on [33]. Protein domains predicted with SMART (http://smart.embl-heidelberg.de/) and PFSCAN (http://hits.isb-sib.ch/cgi-bin/PFSCAN) include GRAM (glucosyltransferase, Rab-like GTPase activator of myotubularins) domain, RID (Rac-induced recruitment domain), PTP/DSP domain, SID (SET-interacting domain), PY motif (XPPXY), CC (coiled-coil) domain and FYVE domain. (B) Western blot of hMTMR4, Nedd4 and tubulin from transfected (MTM tx) or untransfected (untx) HEK-293 lysate immunoprecipitated with anti-MTMR4 antibodies (MTM I.P.). Equal amounts of whole lysate were run as a control. (C) Lysates (lysate) and immunoprecipitates using anti-Nedd4 antibodies (Nedd4 I.P.) or anti-(p70 S6 kinase) antibodies (p70 S6 I.P.) as negative control, of HeLa cells transfected with HA–hMTMR4 wild-type (MTM WT), HA–hMTMR4 PY mutant (MTM PY), pcDNA 3.1 vector alone (vec) or untransfected (untx). Immunoblotting (IB) was performed with anti-HA (monoclonal) antibodies followed by anti-Nedd4 (polyclonal) antibodies and blots were stripped and re-probed with anti-tubulin antibodies as control. Molecular masses are indicated in kDa.
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Figure 1: MTMR4 is a PY-motif-containing protein expressed in muscle that interacts with Nedd4(A) Schematic diagram of human MTMR4, based on [33]. Protein domains predicted with SMART (http://smart.embl-heidelberg.de/) and PFSCAN (http://hits.isb-sib.ch/cgi-bin/PFSCAN) include GRAM (glucosyltransferase, Rab-like GTPase activator of myotubularins) domain, RID (Rac-induced recruitment domain), PTP/DSP domain, SID (SET-interacting domain), PY motif (XPPXY), CC (coiled-coil) domain and FYVE domain. (B) Western blot of hMTMR4, Nedd4 and tubulin from transfected (MTM tx) or untransfected (untx) HEK-293 lysate immunoprecipitated with anti-MTMR4 antibodies (MTM I.P.). Equal amounts of whole lysate were run as a control. (C) Lysates (lysate) and immunoprecipitates using anti-Nedd4 antibodies (Nedd4 I.P.) or anti-(p70 S6 kinase) antibodies (p70 S6 I.P.) as negative control, of HeLa cells transfected with HA–hMTMR4 wild-type (MTM WT), HA–hMTMR4 PY mutant (MTM PY), pcDNA 3.1 vector alone (vec) or untransfected (untx). Immunoblotting (IB) was performed with anti-HA (monoclonal) antibodies followed by anti-Nedd4 (polyclonal) antibodies and blots were stripped and re-probed with anti-tubulin antibodies as control. Molecular masses are indicated in kDa.
Mentions: As Nedd4 overexpression was correlated with the progression of atrophic processes in muscle cells, we sought potential substrates in muscle that may be targeted by Nedd4 to elicit its effects. To this end, we took a bioinformatics approach to elucidate PY-motif-containing proteins that were expressed in skeletal muscle. Nedd4 consists of an N-terminal C2 domain, three or four WW domains and the ubiquitin ligase HECT domain and, in many cases, engages in interactions with its substrates via a WW domain–PY motif interaction [4–9]. Searching a SIDNET (Signalling Identification Network) MGC (Mammalian Gene Collection) clone database (http://www.sickkids.ca/Research/SIDNET/SIDNET.html) for PY-motif-containing proteins that were expressed in skeletal muscle limited potential substrates of Nedd4 to several proteins, including MTMR4, which belongs to a family of lipid tyrosine phosphatases with specificity towards PtdIns3P and PtdIns(3,5)P2 [14,15]. MTMR4 possesses several protein–protein and protein–lipid interaction domains (Figure 1A) including the FYVE domain, shown to mediate its localization to endosomes through its interaction with PtdIns3P and PtdIns(3,5)P2 [14] and is the only family member possessing a PY motif. The MTMR4 PY-motif (VPPLY) at the C-terminus (positions 1004–1008) is conserved across mammalian species (results not shown), adhering to the canonical L/XPPXY consensus sequence for WW domain type I binding. To verify that Nedd4 and MTMR4 can interact, cDNA for hMTMR4 was transfected into HEK-293 cells, and lysate from these cells was subjected to immunoprecipitation with anti-MTMR4 antibodies. Immunoprecipitates were separated by SDS/PAGE and probed with anti-MTMR4 and anti-Nedd4 antibodies to determine whether the two proteins interacted, with anti-tubulin antibodies used as a control. Immunoprecipitated transfected hMTMR4 interacted with endogenous Nedd4, but no band was detected in untransfected lysate (Figure 1B). Similarly, cell lysate transfected with HA-tagged hMTMR4 wild-type and PY-motif mutant was immunoprecipitated with anti-Nedd4 antibodies, and subjected to Western blotting with anti-Nedd4, anti-HA and anti-tubulin antibodies. Endogenous Nedd4 was able to immunoprecipitate transfected wild-type hMTMR4, but not the MTMR4 PY mutant, suggesting that the endogenous association of the two proteins is likely to be mediated through the WW domain–PY motif interaction (Figure 1C).

Bottom Line: The inositol phosphatase, MTMR4 (myotubularin-related protein 4), was identified as a novel interactor of the ubiquitin ligase Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4). hMTMR4 (human MTMR4) and Nedd4 co-immunoprecipitated and co-localized to late endosomes.The PY (Pro-Tyr) motif of hMTMR4 binds to WW (Trp-Trp) domains of hNedd4.MTMR4 expression was decreased in atrophying muscle, whereas Nedd4 expression was increased and hMTMR4 was ubiquitinated by hNedd4, suggesting that this novel interaction may underlie the biological process of muscle breakdown.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Clinical Sciences Division, Room 7344, Medical Science Building, University of Toronto, Toronto, 1 Kings College Circle, Ontario, Canada.

ABSTRACT
The inositol phosphatase, MTMR4 (myotubularin-related protein 4), was identified as a novel interactor of the ubiquitin ligase Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4). hMTMR4 (human MTMR4) and Nedd4 co-immunoprecipitated and co-localized to late endosomes. The PY (Pro-Tyr) motif of hMTMR4 binds to WW (Trp-Trp) domains of hNedd4. MTMR4 expression was decreased in atrophying muscle, whereas Nedd4 expression was increased and hMTMR4 was ubiquitinated by hNedd4, suggesting that this novel interaction may underlie the biological process of muscle breakdown.

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