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Article ; Online: An In Vitro Kinase Assay to Assess Rac1 Phosphorylation by ERK.

Brandwein, Daniel / Tong, Junfeng / Li, Laiji / Ballermann, Barbara / Wang, Zhixiang

Methods in molecular biology (Clifton, N.J.)

2018 Volume 1821, Page(s) 131–140

Abstract: Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that T108 within ... ...

Abstract	Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that T108 within the
MeSH term(s)	Adenosine Triphosphate/chemistry ; Amino Acid Motifs ; Extracellular Signal-Regulated MAP Kinases/chemistry ; Extracellular Signal-Regulated MAP Kinases/genetics ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Isotope Labeling/methods ; Phosphorylation ; Recombinant Fusion Proteins/chemistry ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/metabolism ; rac1 GTP-Binding Protein/chemistry ; rac1 GTP-Binding Protein/genetics ; rac1 GTP-Binding Protein/metabolism
Chemical Substances	RAC1 protein, human ; Recombinant Fusion Proteins ; Adenosine Triphosphate (8L70Q75FXE) ; Extracellular Signal-Regulated MAP Kinases (EC 2.7.11.24) ; rac1 GTP-Binding Protein (EC 3.6.5.2)
Language	English
Publishing date	2018-07-31
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-8612-5_9
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Article ; Online: Phosphorylation and Activation of RhoA by ERK in Response to Epidermal Growth Factor Stimulation.

Tong, Junfeng / Li, Laiji / Ballermann, Barbara / Wang, Zhixiang

PloS one

2016 Volume 11, Issue 1, Page(s) e0147103

Abstract: The small GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, cell motility, and cytokinesis. In addition to the canonical GTPase cycle, recent findings have suggested that phosphorylation further ... ...

Abstract	The small GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, cell motility, and cytokinesis. In addition to the canonical GTPase cycle, recent findings have suggested that phosphorylation further contributes to the tight regulation of Rho GTPases. Indeed, RhoA is phosphorylated on serine 188 (188S) by a number of protein kinases. We have recently reported that Rac1 is phosphorylated on threonine 108 (108T) by extracellular signal-regulated kinases (ERK) in response to epidermal growth factor (EGF) stimulation. Here, we provide evidence that RhoA is phosphorylated by ERK on 88S and 100T in response to EGF stimulation. We show that ERK interacts with RhoA and that this interaction is dependent on the ERK docking site (D-site) at the C-terminus of RhoA. EGF stimulation enhanced the activation of the endogenous RhoA. The phosphomimetic mutant, GFP-RhoA S88E/T100E, when transiently expressed in COS-7 cells, displayed higher GTP-binding than wild type RhoA. Moreover, the expression of GFP-RhoA S88E/T100E increased actin stress fiber formation in COS-7 cells, which is consistent with its higher activity. In contrast to Rac1, phosphorylation of RhoA by ERK does not target RhoA to the nucleus. Finally, we show that regardless of the phosphorylation status of RhoA and Rac1, substitution of the RhoA PBR with the Rac1 PBR targets RhoA to the nucleus and substitution of Rac1 PBR with RhoA PBR significantly reduces the nuclear localization of Rac1. In conclusion, ERK phosphorylates RhoA on 88S and 100T in response to EGF, which upregulates RhoA activity.
MeSH term(s)	Actins/metabolism ; Animals ; COS Cells ; Cell Line ; Cell Nucleus/metabolism ; Cercopithecus aethiops ; Cytoskeleton/metabolism ; Epidermal Growth Factor/pharmacology ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Phosphorylation/drug effects ; Signal Transduction/drug effects ; rhoA GTP-Binding Protein/metabolism
Chemical Substances	Actins ; Epidermal Growth Factor (62229-50-9) ; Extracellular Signal-Regulated MAP Kinases (EC 2.7.11.24) ; rhoA GTP-Binding Protein (EC 3.6.5.2)
Language	English
Publishing date	2016-01-27
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0147103
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Article ; Online: TIMAP promotes angiogenesis by suppressing PTEN-mediated Akt inhibition in human glomerular endothelial cells.

Obeidat, Marya / Li, Laiji / Ballermann, Barbara J

American journal of physiology. Renal physiology

2014 Volume 307, Issue 5, Page(s) F623–33

Abstract: The function of TIMAP, an endothelial cell (EC)-predominant protein phosphatase 1-regulatory subunit, is poorly understood. We explored the potential role of TIMAP in the Akt-dependent regulation of glomerular EC proliferation, survival, and in vitro ... ...

Abstract	The function of TIMAP, an endothelial cell (EC)-predominant protein phosphatase 1-regulatory subunit, is poorly understood. We explored the potential role of TIMAP in the Akt-dependent regulation of glomerular EC proliferation, survival, and in vitro angiogenesis. To deplete TIMAP, the EC were transfected with TIMAP-specific or nonspecific small interfering (si) RNA. The rate of electrical impedance development across subconfluent EC monolayers, a measure of the time-dependent increase in EC number, was 93 ± 2% lower in TIMAP-depleted than in control EC. This effect on cell proliferation was associated with reduced DNA synthesis and increased apoptosis: TIMAP silencing reduced 5-ethynyl-2'-deoxyuridine incorporation by 38 ± 2% during the exponential phase of EC proliferation, and cleaved caspase 3 as well as caspase 3 activity increased in TIMAP-depleted relative to control cells. Furthermore, TIMAP depletion inhibited the formation of angiogenic sprouts by glomerular EC in three-dimensional culture. TIMAP depletion strongly diminished growth factor-stimulated Akt phosphorylation without altering ERK1/2 phosphorylation, suggesting a specific effect on the PI3K/Akt/PTEN pathway. Endogenous TIMAP and PTEN colocalized in EC and coimmunoprecipitated from EC lysates. The inhibitory PTEN phosphorylation on S370 was significantly reduced in TIMAP-depleted compared with control EC, while phosphorylation of PTEN on the S380/T382/T383 cluster remained unchanged. Finally, the PTEN inhibitor bpV(phen) fully reversed the suppressive effect of TIMAP depletion on Akt phosphorylation. The data indicate that in growing EC, TIMAP is necessary for Akt-dependent EC proliferation, survival, and angiogenic sprout formation and that this effect of TIMAP is mediated by inhibition of the tumor suppressor PTEN.
MeSH term(s)	Apoptosis/physiology ; Cell Proliferation/physiology ; Cell Survival/physiology ; Cells, Cultured ; Endothelial Cells/cytology ; Endothelial Cells/physiology ; Humans ; In Vitro Techniques ; Kidney Glomerulus/cytology ; Kidney Glomerulus/physiology ; Membrane Proteins/physiology ; Neovascularization, Physiologic/physiology ; PTEN Phosphohydrolase/antagonists & inhibitors ; PTEN Phosphohydrolase/physiology ; Phosphatidylinositol 3-Kinases/physiology ; Phosphorylation/physiology ; Proto-Oncogene Proteins c-akt/antagonists & inhibitors ; Proto-Oncogene Proteins c-akt/physiology
Chemical Substances	Membrane Proteins ; PPP1R16B protein, human ; Phosphatidylinositol 3-Kinases (EC 2.7.1.-) ; Proto-Oncogene Proteins c-akt (EC 2.7.11.1) ; PTEN Phosphohydrolase (EC 3.1.3.67) ; PTEN protein, human (EC 3.1.3.67)
Language	English
Publishing date	2014-09-01
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	603837-2
ISSN	1522-1466 ; 0363-6127
ISSN (online)	1522-1466
ISSN	0363-6127
DOI	10.1152/ajprenal.00070.2014
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Article ; Online: TIMAP inhibits endothelial myosin light chain phosphatase by competing with MYPT1 for the catalytic protein phosphatase 1 subunit PP1cβ.

Wang, Xin / Obeidat, Marya / Li, Laiji / Pasarj, Phuwadet / Aburahess, Salah / Holmes, Charles F B / Ballermann, Barbara J

The Journal of biological chemistry

2019 Volume 294, Issue 36, Page(s) 13280–13291

Abstract: Transforming growth factor-β membrane associated protein (TIMAP) is an endothelial cell (EC)-predominant PP1 regulatory subunit and a member of the myosin phosphatase target (MYPT) protein family. The MYPTs preferentially bind the catalytic protein ... ...

Abstract	Transforming growth factor-β membrane associated protein (TIMAP) is an endothelial cell (EC)-predominant PP1 regulatory subunit and a member of the myosin phosphatase target (MYPT) protein family. The MYPTs preferentially bind the catalytic protein phosphatase 1 subunit PP1cβ, forming myosin phosphatase holoenzymes. We investigated whether TIMAP/PP1cβ could also function as a myosin phosphatase. Endogenous PP1cβ, myosin light chain 2 (MLC2), and myosin IIA heavy chain coimmunoprecipitated from EC lysates with endogenous TIMAP, and endogenous MLC2 colocalized with TIMAP in EC projections. Purified recombinant GST-TIMAP interacted directly with purified recombinant His-MLC2. However, TIMAP overexpression in EC enhanced MLC2 phosphorylation, an effect not observed with a TIMAP mutant that does not bind PP1cβ. Conversely, MLC2 phosphorylation was reduced in lung lysates from TIMAP-deficient mice and upon silencing of endogenous TIMAP expression in ECs. Ectopically expressed TIMAP slowed the rate of MLC2 dephosphorylation, an effect requiring TIMAP-PP1cβ interaction. The association of MYPT1 with PP1cβ was profoundly reduced in the presence of excess TIMAP, leading to proteasomal MYPT1 degradation. In the absence of TIMAP, MYPT1-associated PP1cβ readily bound immobilized microcystin-LR, an active-site inhibitor of PP1c. By contrast, TIMAP-associated PP1cβ did not interact with microcystin-LR, indicating that the active site of PP1cβ is blocked when it is bound to TIMAP. Thus, TIMAP inhibits myosin phosphatase activity in ECs by competing with MYPT1 for PP1cβ and blocking the PP1cβ active site.
MeSH term(s)	Animals ; Biocatalysis ; Cell Line ; Endothelial Cells/metabolism ; Humans ; Membrane Proteins/metabolism ; Mice ; Mice, Inbred C57BL ; Myosin-Light-Chain Phosphatase/antagonists & inhibitors ; Myosin-Light-Chain Phosphatase/metabolism ; Protein Phosphatase 1/metabolism
Chemical Substances	Membrane Proteins ; PPP1R16B protein, human ; Protein Phosphatase 1 (EC 3.1.3.16) ; Myosin-Light-Chain Phosphatase (EC 3.1.3.53) ; PPP1R12A protein, human (EC 3.1.3.53)
Language	English
Publishing date	2019-07-17
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.RA118.006075
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Article ; Online: Phosphorylation of Rac1 T108 by extracellular signal-regulated kinase in response to epidermal growth factor: a novel mechanism to regulate Rac1 function.

Tong, Junfeng / Li, Laiji / Ballermann, Barbara / Wang, Zhixiang

Molecular and cellular biology

2013 Volume 33, Issue 22, Page(s) 4538–4551

Abstract: Accumulating evidence has implicated Rho GTPases, including Rac1, in many aspects of cancer development. Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of ... ...

Abstract	Accumulating evidence has implicated Rho GTPases, including Rac1, in many aspects of cancer development. Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that Rac1 T108 within the (106)PNTP(109) motif is likely an extracellular signal-regulated kinase (ERK) phosphorylation site and that Rac1 also has an ERK docking site, (183)KKRKRKCLLL(192) (D site), at the C terminus. Indeed, we show here that both transfected and endogenous Rac1 interacts with ERK and that this interaction is mediated by its D site. Green fluorescent protein (GFP)-Rac1 is threonine (T) phosphorylated in response to epidermal growth factor (EGF), and EGF-induced Rac1 threonine phosphorylation is dependent on the activation of ERK. Moreover, mutant Rac1 with the mutation of T108 to alanine (A) is not threonine phosphorylated in response to EGF. In vitro ERK kinase assay further shows that pure active ERK phosphorylates purified Rac1 but not mutant Rac1 T108A. We also show that Rac1 T108 phosphorylation decreases Rac1 activity, partially due to inhibiting its interaction with phospholipase C-γ1 (PLC-γ1). T108 phosphorylation targets Rac1 to the nucleus, which isolates Rac1 from other guanine nucleotide exchange factors (GEFs) and hinders Rac1's role in cell migration. We conclude that Rac1 T108 is phosphorylated by ERK in response to EGF, which plays an important role in regulating Rac1.
MeSH term(s)	Active Transport, Cell Nucleus ; Amino Acid Sequence ; Animals ; COS Cells ; Cell Line, Tumor ; Cell Movement ; Chlorocebus aethiops ; Enzyme Activation ; Epidermal Growth Factor/metabolism ; Extracellular Signal-Regulated MAP Kinases/chemistry ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Molecular Sequence Data ; Phospholipase C gamma/metabolism ; Phosphorylation ; Protein Interaction Maps ; rac1 GTP-Binding Protein/chemistry ; rac1 GTP-Binding Protein/metabolism
Chemical Substances	Epidermal Growth Factor (62229-50-9) ; Extracellular Signal-Regulated MAP Kinases (EC 2.7.11.24) ; Phospholipase C gamma (EC 3.1.4.3) ; rac1 GTP-Binding Protein (EC 3.6.5.2)
Language	English
Publishing date	2013-09-16
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	779397-2
ISSN	1098-5549 ; 0270-7306
ISSN (online)	1098-5549
ISSN	0270-7306
DOI	10.1128/MCB.00822-13
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Zs.A 1666: Show issues

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Article ; Online: Phosphorylation of Rac1 T108 by Extracellular Signal-Regulated Kinase in Response to Epidermal Growth Factor: a Novel Mechanism To Regulate Rac1 Function

Tong, Junfeng / Li, Laiji / Ballermann, Barbara / Wang, Zhixiang

Molecular and Cellular Biology. 2013 Nov. 1, v. 33, no. 22 p.4538-4551

2013

Abstract	Accumulating evidence has implicated Rho GTPases, including Rac1, in many aspects of cancer development. Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that Rac1 T108 within the ¹⁰⁶PNTP¹⁰⁹ motif is likely an extracellular signal-regulated kinase (ERK) phosphorylation site and that Rac1 also has an ERK docking site, ¹⁸³KKRKRKCLLL¹⁹² (D site), at the C terminus. Indeed, we show here that both transfected and endogenous Rac1 interacts with ERK and that this interaction is mediated by its D site. Green fluorescent protein (GFP)-Rac1 is threonine (T) phosphorylated in response to epidermal growth factor (EGF), and EGF-induced Rac1 threonine phosphorylation is dependent on the activation of ERK. Moreover, mutant Rac1 with the mutation of T108 to alanine (A) is not threonine phosphorylated in response to EGF. In vitro ERK kinase assay further shows that pure active ERK phosphorylates purified Rac1 but not mutant Rac1 T108A. We also show that Rac1 T108 phosphorylation decreases Rac1 activity, partially due to inhibiting its interaction with phospholipase C-γ1 (PLC-γ1). T108 phosphorylation targets Rac1 to the nucleus, which isolates Rac1 from other guanine nucleotide exchange factors (GEFs) and hinders Rac1's role in cell migration. We conclude that Rac1 T108 is phosphorylated by ERK in response to EGF, which plays an important role in regulating Rac1.
Keywords	alanine ; carcinogenesis ; cell movement ; epidermal growth factor ; green fluorescent protein ; guanosinetriphosphatase ; mitogen-activated protein kinase ; mutants ; mutation ; phospholipases ; phosphorylation ; sequence analysis ; threonine
Language	English
Dates of publication	2013-1101
Size	p. 4538-4551.
Publishing place	Taylor & Francis
Document type	Article ; Online
ZDB-ID	779397-2
ISSN	1098-5549 ; 0270-7306
ISSN (online)	1098-5549
ISSN	0270-7306
DOI	10.1128/MCB.00822-13
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Clustered PI(4,5)P₂ accumulation and ezrin phosphorylation in response to CLIC5A.

Al-Momany, Abass / Li, Laiji / Alexander, R Todd / Ballermann, Barbara J

Journal of cell science

2014 Volume 127, Issue Pt 24, Page(s) 5164–5178

Abstract: CLIC5A (encoded by CLIC5) is a component of the ezrin-NHERF2-podocalyxin complex in renal glomerular podocyte foot processes. We explored the mechanism(s) by which CLIC5A regulates ezrin function. In COS-7 cells, CLIC5A augmented ezrin phosphorylation ... ...

Abstract	CLIC5A (encoded by CLIC5) is a component of the ezrin-NHERF2-podocalyxin complex in renal glomerular podocyte foot processes. We explored the mechanism(s) by which CLIC5A regulates ezrin function. In COS-7 cells, CLIC5A augmented ezrin phosphorylation without changing ezrin abundance, increased the association of ezrin with the cytoskeletal fraction and enhanced actin polymerization and the formation of cell surface projections. CLIC5A caused the phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] reporter RFP-PH-PLC to translocate from the cytosol to discrete plasma membrane clusters at the cell surface, where it colocalized with CLIC5A. Transiently expressed HA-PIP5Kα colocalized with GFP-CLIC5A and was pulled from cell lysates by GST-CLIC5A, and silencing of endogenous PIP5Kα abrogated CLIC5A-dependent ERM phosphorylation. N- and C-terminal deletion mutants of CLIC5A, which failed to associate with the plasma membrane, failed to colocalize with PIP5Kα, did not alter the abundance of PI(4,5)P2 plasma membrane clusters and failed to enhance ezrin phosphorylation. Relative to wild-type mice, in CLIC5-deficient mice, the phosphorylation of glomerular ezrin was diminished and the cytoskeletal association of both ezrin and NHERF2 was reduced. Therefore, the mechanism of CLIC5A action involves clustered plasma membrane PI(4,5)P2 accumulation through an interaction of CLIC5A with PI(4,5)P2-generating kinases, in turn facilitating ezrin activation and actin-dependent cell surface remodeling.
MeSH term(s)	Actins/metabolism ; Animals ; COS Cells ; Cell Membrane/drug effects ; Cell Membrane/metabolism ; Chloride Channels/metabolism ; Chlorocebus aethiops ; Cytoskeletal Proteins/metabolism ; Gene Silencing/drug effects ; Glycolates/pharmacology ; HeLa Cells ; Humans ; Kidney Glomerulus/drug effects ; Kidney Glomerulus/metabolism ; Membrane Proteins/metabolism ; Mice ; Microfilament Proteins/metabolism ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Phosphorylation/drug effects ; Phosphotransferases (Alcohol Group Acceptor)/metabolism ; Polymerization/drug effects ; Sialoglycoproteins/metabolism ; Sulfonamides/pharmacology ; Transfection ; Type C Phospholipases/metabolism
Chemical Substances	2,4,6-trimethyl-N-(meta-3-trifluoromethylphenyl)benzenesulfonamide ; Actins ; CLIC5 protein, human ; CLIC5 protein, mouse ; Chloride Channels ; Cytoskeletal Proteins ; Glycolates ; Membrane Proteins ; Microfilament Proteins ; Phosphatidylinositol 4,5-Diphosphate ; Sialoglycoproteins ; Sulfonamides ; ezrin ; podocalyxin ; moesin (144131-77-1) ; radixin (144517-21-5) ; MK 473 (54197-05-6) ; Phosphotransferases (Alcohol Group Acceptor) (EC 2.7.1.-) ; 1-phosphatidylinositol-4-phosphate 5-kinase (EC 2.7.1.68) ; Type C Phospholipases (EC 3.1.4.-)
Language	English
Publishing date	2014-10-24
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2993-2
ISSN	1477-9137 ; 0021-9533
ISSN (online)	1477-9137
ISSN	0021-9533
DOI	10.1242/jcs.147744
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Article ; Online: Both CLIC4 and CLIC5A activate ERM proteins in glomerular endothelium.

Tavasoli, Mahtab / Al-Momany, Abass / Wang, Xin / Li, Laiji / Edwards, John C / Ballermann, Barbara J

American journal of physiology. Renal physiology

2016 Volume 311, Issue 5, Page(s) F945–F957

Abstract: The chloride intracellular channel (CLIC) 5A is expressed at very high levels in renal glomeruli, in both endothelial cells (EC) and podocytes. CLIC5A stimulates Rac1- and phosphatidylinositol (4,5)-bisphosphate-dependent ERM (ezrin, radixin, moesin) ... ...

Abstract	The chloride intracellular channel (CLIC) 5A is expressed at very high levels in renal glomeruli, in both endothelial cells (EC) and podocytes. CLIC5A stimulates Rac1- and phosphatidylinositol (4,5)-bisphosphate-dependent ERM (ezrin, radixin, moesin) activation. ERM proteins, in turn, function in lumen formation and in the development of actin-based cellular projections. In mice lacking CLIC5A, ERM phosphorylation is profoundly reduced in podocytes, but preserved in glomerular EC. Since glomerular EC also express CLIC4, we reasoned that, if CLIC4 activates ERM proteins like CLIC5A, then CLIC4 could compensate for the CLIC5A loss in glomerular EC. In glomeruli of CLIC5-deficient mice, CLIC4 expression was upregulated and colocalized with moesin and ezrin in glomerular EC, but not in podocytes. In cultured glomerular EC, CLIC4 silencing reduced ERM phosphorylation and cytoskeletal association, and expression of exogenous CLIC4 or CLIC5A rescued ERM de-phosphorylation due to CLIC4 silencing. In mice lacking either CLIC4 or CLIC5, ERM phosphorylation was retained in glomerular EC, but, in mice lacking both CLIC4 and CLIC5, glomerular EC ERM phosphorylation was profoundly reduced. Although glomerular EC fenestrae developed normally in dual CLIC4/CLIC5-deficient mice, the density of fenestrae declined substantially by 8 mo of age, along with the deposition of subendothelial electron-lucent material. The dual CLIC4/CLIC5-deficient mice developed spontaneous proteinuria, glomerular cell proliferation, and matrix deposition. Thus CLIC4 stimulates ERM activation and can compensate for CLIC5A in glomerular EC. The findings indicate that CLIC4/CLIC5A-mediated ERM activation is required for maintenance of the glomerular capillary architecture.
MeSH term(s)	Animals ; Chloride Channels/genetics ; Chloride Channels/metabolism ; Cytoskeletal Proteins/metabolism ; Endothelial Cells/cytology ; Endothelial Cells/metabolism ; Kidney Glomerulus/cytology ; Kidney Glomerulus/metabolism ; Membrane Proteins/metabolism ; Mice ; Mice, Knockout ; Microfilament Proteins/metabolism ; Mitochondrial Proteins/genetics ; Mitochondrial Proteins/metabolism ; Phosphorylation ; Podocytes/metabolism
Chemical Substances	CLIC protein, mouse ; CLIC5 protein, mouse ; Chloride Channels ; Cytoskeletal Proteins ; Membrane Proteins ; Microfilament Proteins ; Mitochondrial Proteins ; ezrin ; moesin (144131-77-1) ; radixin (144517-21-5)
Language	English
Publishing date	2016-11-01
Publishing country	United States
Document type	Journal Article
ZDB-ID	603837-2
ISSN	1522-1466 ; 0363-6127
ISSN (online)	1522-1466
ISSN	0363-6127
DOI	10.1152/ajprenal.00353.2016
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Uc I Zs.89: Show issues

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Article ; Online: The chloride intracellular channel 5A stimulates podocyte Rac1, protecting against hypertension-induced glomerular injury.

Tavasoli, Mahtab / Li, Laiji / Al-Momany, Abass / Zhu, Lin-Fu / Adam, Benjamin A / Wang, Zhixiang / Ballermann, Barbara J

Kidney international

2016 Volume 89, Issue 4, Page(s) 833–847

Abstract: Glomerular capillary hypertension elicits podocyte remodeling and is a risk factor for the progression of glomerular disease. Ezrin, which links podocalyxin to actin in podocytes, is activated through the chloride intracellular channel 5A (CLIC5A)- ... ...

Abstract	Glomerular capillary hypertension elicits podocyte remodeling and is a risk factor for the progression of glomerular disease. Ezrin, which links podocalyxin to actin in podocytes, is activated through the chloride intracellular channel 5A (CLIC5A)-dependent phosphatidylinositol 4,5 bisphosphate (PI[4,5]P2) accumulation. Because Rac1 is involved in podocyte actin remodeling and can promote PI[4,5]P2 production we determined whether CLIC5A-dependent PI[4,5]P2 generation and ezrin activation are mediated by Rac1. In COS7 cells, CLIC5A expression stimulated Rac1 but not Cdc42 or Rho activity. CLIC5A also stimulated phosphorylation of the Rac1 effector Pak1 in COS7 cells and in cultured mouse podocytes. CLIC5A-induced PI[4,5]P2 accumulation and Pak1 and ezrin phosphorylation were all Rac1 dependent. In DOCA/Salt hypertension, phosphorylated Pak increased in podocytes of wild-type, but not CLIC5-deficient mice. In DOCA/salt hypertensive mice lacking CLIC5, glomerular capillary microaneurysms were more frequent and albuminuria was greater than in wild-type mice. Thus, augmented hypertension-induced glomerular capillary injury in mice lacking CLIC5 results from abrogation of Rac1-dependent Pak and ezrin activation, perhaps reducing the tensile strength of the podocyte actin cytoskeleton.
MeSH term(s)	Animals ; COS Cells ; Cercopithecus aethiops ; Chloride Channels/metabolism ; Cytoskeletal Proteins/metabolism ; Disease Models, Animal ; Female ; Hypertension/complications ; Kidney Diseases/etiology ; Kidney Diseases/metabolism ; Male ; Mice ; Microfilament Proteins/metabolism ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Phosphorylation ; Podocytes/metabolism ; Sialoglycoproteins/metabolism ; cdc42 GTP-Binding Protein/metabolism ; p21-Activated Kinases/metabolism ; rac1 GTP-Binding Protein/metabolism ; rho-Associated Kinases/metabolism
Chemical Substances	CLIC5 protein, human ; Chloride Channels ; Cytoskeletal Proteins ; Microfilament Proteins ; Phosphatidylinositol 4,5-Diphosphate ; Sialoglycoproteins ; podocalyxin ; p21-Activated Kinases (EC 2.7.11.1) ; rho-Associated Kinases (EC 2.7.11.1) ; cdc42 GTP-Binding Protein (EC 3.6.5.2) ; rac1 GTP-Binding Protein (EC 3.6.5.2)
Language	English
Publishing date	2016-04
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	120573-0
ISSN	1523-1755 ; 0085-2538
ISSN (online)	1523-1755
ISSN	0085-2538
DOI	10.1016/j.kint.2016.01.001
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Zs.A 797: Show issues

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Article: Multi-directional function of the protein phosphatase 1 regulatory subunit TIMAP

Shopik, Micheal J / Li, Laiji / Luu, Hue-Anh / Obeidat, Marya / Holmes, Charles F.B / Ballermann, Barbara J

Biochemical and biophysical research communications. 2013 June 14, v. 435, no. 4

2013

Abstract: TIMAP is an endothelial-cell predominant member of the MYPT family of PP1c regulatory subunits. This study explored the TIMAP–PP1c interaction and substrate specificity in vitro. TIMAP associated with all three PP1c isoforms, but endogenous endothelial ... ...

Abstract	TIMAP is an endothelial-cell predominant member of the MYPT family of PP1c regulatory subunits. This study explored the TIMAP–PP1c interaction and substrate specificity in vitro. TIMAP associated with all three PP1c isoforms, but endogenous endothelial cell TIMAP preferentially co-immunoprecipitated with PP1cβ. Structural modeling of the TIMAP/PP1c complex predicts that the PP1c C-terminus is buried in the TIMAP ankyrin cluster, and that the PP1c active site remains accessible. Consistent with this model, C-terminal PP1c phosphorylation by cdk2-cyclinA was masked by TIMAP, and PP1c bound TIMAP when the active site was occupied by the inhibitor microcystin. TIMAP inhibited PP1c activity toward phosphorylase a in a concentration-dependent manner, with half-maximal inhibition in the 0.4–1.2nM range, an effect modulated by the length, and by Ser333/Ser337 phosphomimic mutations of the TIMAP C-terminus. TIMAP-bound PP1cβ effectively dephosphorylated MLC2 and TIMAP itself. By contrast, TIMAP inhibited the PP1cβ activity toward the putative substrate LAMR1, and instead masked LAMR1 PKA- and PKC-phosphorylation sites. This is direct evidence that MLC2 is a TIMAP/PP1c substrate. The data also indicate that TIMAP can modify protein phosphorylation independent of its function as a PP1c regulatory subunit, namely by masking phosphorylation sites of binding partners like PP1c and LAMR1.
Keywords	active sites ; binding sites ; cAMP-dependent protein kinase ; endothelial cells ; microcystins ; models ; mutation ; phosphorylase ; protein phosphorylation ; substrate specificity
Language	English
Dates of publication	2013-0614
Size	p. 567-573.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	205723-2
ISSN	0006-291X ; 0006-291X
ISSN (online)	0006-291X
ISSN	0006-291X
DOI	10.1016/j.bbrc.2013.05.012
Database	NAL-Catalogue (AGRICOLA)

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