Browsing by Subject "Phosphoproteins"

Now showing 1 - 20 of 21

Open Access
A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins.
(Proc Natl Acad Sci U S A, 1998-07-21) Hall, RA; Ostedgaard, LS; Premont, RT; Blitzer, JT; Rahman, N; Welsh, MJ; Lefkowitz, RJ
The Na+/H+ exchanger regulatory factor (NHERF) binds to the tail of the beta2-adrenergic receptor and plays a role in adrenergic regulation of Na+/H+ exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the beta2 receptor. Mutagenesis studies of the beta2 receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S/T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the beta2 receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.
Open Access
Age-related changes in the cellular composition and epithelial organization of the mouse trachea.
(PloS one, 2014-01) Wansleeben, Carolien; Bowie, Emily; Hotten, Danielle F; Yu, Yen-Rei A; Hogan, Brigid LM
We report here senescent changes in the structure and organization of the mucociliary pseudostratified epithelium of the mouse trachea and main stem bronchi. We confirm previous reports of the gradual appearance of age-related, gland-like structures (ARGLS) in the submucosa, especially in the intercartilage regions and carina. Immunohistochemistry shows these structures contain ciliated and secretory cells and Krt5+ basal cells, but not the myoepithelial cells or ciliated ducts typical of normal submucosal glands. Data suggest they arise de novo by budding from the surface epithelium rather than by delayed growth of rudimentary or cryptic submucosal glands. In old mice the surface epithelium contains fewer cells per unit length than in young mice and the proportion of Krt5+, p63+ basal cells is reduced in both males and females. However, there appears to be no significant difference in the ability of basal stem cells isolated from individual young and old mice to form clonal tracheospheres in culture or in the ability of the epithelium to repair after damage by inhaled sulfur dioxide. Gene expression analysis by Affymetrix microarray and quantitative PCR, as well as immunohistochemistry and flow sorting studies, are consistent with low-grade chronic inflammation in the tracheas of old versus young mice and an increase in the number of immune cells. The significance of these changes for ARGL formation are not clear since several treatments that induce acute inflammation in young mice did not result in budding of the surface epithelium.
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beta-arrestin-1 competitively inhibits insulin-induced ubiquitination and degradation of insulin receptor substrate 1.
(Mol Cell Biol, 2004-10) Usui, Isao; Imamura, Takeshi; Huang, Jie; Satoh, Hiroaki; Shenoy, Sudha K; Lefkowitz, Robert J; Hupfeld, Christopher J; Olefsky, Jerrold M
beta-arrestin-1 is an adaptor protein that mediates agonist-dependent internalization and desensitization of G-protein-coupled receptors (GPCRs) and also participates in the process of heterologous desensitization between receptor tyrosine kinases and GPCR signaling. In the present study, we determined whether beta-arrestin-1 is involved in insulin-induced insulin receptor substrate 1 (IRS-1) degradation. Overexpression of wild-type (WT) beta-arrestin-1 attenuated insulin-induced degradation of IRS-1, leading to increased insulin signaling downstream of IRS-1. When endogenous beta-arrestin-1 was knocked down by transfection of beta-arrestin-1 small interfering RNA, insulin-induced IRS-1 degradation was enhanced. Insulin stimulated the association of IRS-1 and Mdm2, an E3 ubiquitin ligase, and this association was inhibited to overexpression of WT beta-arrestin-1, which led by decreased ubiquitin content of IRS-1, suggesting that both beta-arrestin-1 and IRS-1 competitively bind to Mdm2. In summary, we have found the following: (i) beta-arrestin-1 can alter insulin signaling by inhibiting insulin-induced proteasomal degradation of IRS-1; (ii) beta-arrestin-1 decreases the rate of ubiquitination of IRS-1 by competitively binding to endogenous Mdm2, an E3 ligase that can ubiquitinate IRS-1; (iii) dephosphorylation of S412 on beta-arrestin and the amino terminus of beta-arrestin-1 are required for this effect of beta-arrestin on IRS-1 degradation; and (iv) inhibition of beta-arrestin-1 leads to enhanced IRS-1 degradation and accentuated cellular insulin resistance.
Open Access
beta-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins.
(Proc Natl Acad Sci U S A, 2001-12-18) Chen, W; Hu, LA; Semenov, MV; Yanagawa, S; Kikuchi, A; Lefkowitz, RJ; Miller, WE
One aspect of the function of the beta-arrestins is to serve as scaffold or adapter molecules coupling G-protein coupled receptors (GPCRs) to signal transduction pathways distinct from traditional second messenger pathways. Here we report the identification of Dishevelled 1 and Dishevelled 2 (Dvl1 and Dvl2) as beta-arrestin1 (betaarr1) interacting proteins. Dvl proteins participate as key intermediates in signal transmission from the seven membrane-spanning Frizzled receptors leading to inhibition of glycogen synthase kinase-3beta (GSK-3beta), stabilization of beta-catenin, and activation of the lymphoid enhancer factor (LEF) transcription factor. We find that phosphorylation of Dvl strongly enhances its interaction with betaarr1, suggesting that regulation of Dvl phosphorylation and subsequent interaction with betaarr1 may play a key role in the activation of the LEF transcription pathway. Because coexpression of the Dvl kinases, CK1epsilon and PAR-1, with Dvl synergistically activates LEF reporter gene activity, we reasoned that coexpression of betaarr1 with Dvl might also affect LEF-dependent gene activation. Interestingly, whereas betaarr1 or Dvl alone leads to low-level stimulation of LEF (2- to 5-fold), coexpression of betaarr1 with either Dvl1 or Dvl2 leads to a synergistic activation of LEF (up to 16-fold). Additional experiments with LiCl as an inhibitor of GSK-3beta kinase activity indicate that the step affected by betaarr1 is upstream of GSK-3beta and most likely at the level of Dvl. These results identify betaarr1 as a regulator of Dvl-dependent LEF transcription and suggest that betaarr1 might serve as an adapter molecule that can couple Frizzled receptors and perhaps other GPCRs to these important transcription pathways.
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beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein.
(Proc Natl Acad Sci U S A, 1998-11-24) Premont, RT; Claing, A; Vitale, N; Freeman, JL; Pitcher, JA; Patton, WA; Moss, J; Vaughan, M; Lefkowitz, RJ
G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP ribosylation factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced beta2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating beta2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.
Open Access
Cooperativity between the Phosphorylation of Thr(95) and Ser(77) of NHERF-1 in the Hormonal Regulation of Renal Phosphate Transport
(JOURNAL OF THE ROYAL SOCIETY INTERFACE, 2013-01-06) Weinman, Edward J; Steplock, Deborah; Zhang, Yinghua; Biswas, Rajatsubhra; Bloch, Robert J; Shenolikar, Shirish
The phosphorylation of the sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) plays a key role in the regulation of renal phosphate transport by parathyroid hormone (PTH) and dopamine. Ser(77) in the first PDZ domain of NHERF-1 is a downstream target of both hormones. The current experiments explore the role of Thr(95), another phosphate acceptor site in the PDZ I domain, on hormone-mediated regulation of phosphate transport in the proximal tubule of the kidney. The substitution of alanine for threonine at position 95 (T95A) significantly decreased the rate and extent of in vitro phosphorylation of Ser(77) by PKC. In NHERF-1-null proximal tubule cells, neither PTH nor dopamine inhibited sodium-dependent phosphate transport. Infection of the cells with adenovirus expressing full-length WT GFP-NHERF-1 increased basal phosphate transport and restored the inhibitory effect of both PTH and dopamine. Infection with full-length NHERF-1 containing a T95A mutation, however, increased basal phosphate transport but not the responsiveness to either hormone. As determined by surface plasmon resonance, the substitution of serine for aspartic acid (S77D) in the PDZ I domain decreased the binding affinity to the sodium-dependent phosphate transporter 2a (Npt2a) as compared with WT PDZ I, but a T95D mutation had no effect on binding. Finally, cellular studies indicated that both PTH and dopamine treatment increased the phosphorylation of Thr(95). These studies indicate a remarkable cooperativity between the phosphorylation of Thr(95) and Ser(77) of NHERF-1 in the hormonal regulation of renal phosphate transport. The phosphorylation of Thr(95) facilitates the phosphorylation of Ser(77). This, in turn, results in the dissociation of NHERF-1 from Npt2a and a decrease in phosphate transport in renal proximal tubule cells.
Open Access
DOK2 inhibits EGFR-mutated lung adenocarcinoma.
(PloS one, 2013-01) Berger, Alice H; Chen, Ming; Morotti, Alessandro; Janas, Justyna A; Niki, Masaru; Bronson, Roderick T; Taylor, Barry S; Ladanyi, Marc; Van Aelst, Linda; Politi, Katerina; Varmus, Harold E; Pandolfi, Pier Paolo
Somatic mutations in the EGFR proto-oncogene occur in ~15% of human lung adenocarcinomas and the importance of EGFR mutations for the initiation and maintenance of lung cancer is well established from mouse models and cancer therapy trials in human lung cancer patients. Recently, we identified DOK2 as a lung adenocarcinoma tumor suppressor gene. Here we show that genomic loss of DOK2 is associated with EGFR mutations in human lung adenocarcinoma, and we hypothesized that loss of DOK2 might therefore cooperate with EGFR mutations to promote lung tumorigenesis. We tested this hypothesis using genetically engineered mouse models and find that loss of Dok2 in the mouse accelerates lung tumorigenesis initiated by oncogenic EGFR, but not that initiated by mutated Kras. Moreover, we find that DOK2 participates in a negative feedback loop that opposes mutated EGFR; EGFR mutation leads to recruitment of DOK2 to EGFR and DOK2-mediated inhibition of downstream activation of RAS. These data identify DOK2 as a tumor suppressor in EGFR-mutant lung adenocarcinoma.
Open Access
Dynamics of PTH-induced disassembly of Npt2a/NHERF-1 complexes in living OK cells.
(American journal of physiology. Renal physiology, 2011-01) Weinman, Edward J; Steplock, Deborah; Shenolikar, Shirish; Blanpied, Thomas A
Parathyroid hormone (PTH) inhibits the reabsorption of phosphate in the renal proximal tubule by disrupting the binding of the sodium-dependent phosphate transporter 2A (Npt2a) to the adapter protein sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), a process initiated by activation of protein kinase C (PKC). To gain additional insights into the dynamic sequence of events, the time course of these responses was studied in living opossum kidney (OK) cells. Using a FRET-based biosensor, we found that PTH activated intracellular PKC within seconds to minutes. In cells expressing GFP-Npt2a and mCherry-NHERF, PTH did not affect the relative abundance of NHERF-1 but there was a significant and time-dependent decrease in the Npt2a/NHERF-1 ratio. The half-time to maximal dissociation was 15 to 20 min. By contrast, PTH had no effect on the fluorescence ratio for GFP-ezrin compared with mCherry-NHERF-1 at the apical surface. These experiments establish that PTH treatment of proximal tubule OK cells leads to rapid activation of PKC with the subsequent dissociation of Npt2a/NHERF-1 complexes. The association of NHERF-1 with Ezrin and their localization at the apical membrane, however, was unperturbed by PTH, thereby enabling the rapid recruitment and membrane reinsertion of Npt2a and other NHERF-1 targets on termination of the hormone response.
Open Access
Fibroblast growth factor-23-mediated inhibition of renal phosphate transport in mice requires sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) and synergizes with parathyroid hormone.
(The Journal of biological chemistry, 2011-10) Weinman, Edward J; Steplock, Deborah; Shenolikar, Shirish; Biswas, Rajatsubhra
Fibroblast growth factor-23 (FGF-23) inhibits sodium-dependent phosphate transport in brush border membrane vesicles derived from hormone-treated kidney slices of the mouse and in mouse proximal tubule cells by processes involving mitogen-activated protein kinase (MAPK) but not protein kinase A (PKA) or protein kinase C (PKC). By contrast, phosphate transport in brush border membrane vesicles and proximal tubule cells from sodium-hydrogen exchanger regulatory factor-1 (NHERF-1)-null mice were resistant to the inhibitory effect of FGF-23 (10(-9) m). Infection of NHERF-1-null proximal tubule cells with wild-type adenovirus-GFP-NHERF-1 increased basal phosphate transport and restored the inhibitory effect of FGF-23. Infection with adenovirus-GFP-NHERF-1 containing a S77A or T95D mutation also increased basal phosphate transport, but the cells remained resistant to FGF-23 (10(-9) m). Low concentrations of FGF-23 (10(-13) m) and PTH (10(-11) m) individually did not inhibit phosphate transport or activate PKA, PKC, or MAPK. When combined, however, these hormones markedly inhibited phosphate transport associated with activation of PKC and PKA but not MAPK. These studies indicate that FGF-23 inhibits phosphate transport in the mouse kidney by processes that involve the scaffold protein NHERF-1. In addition, FGF-23 synergizes with PTH to inhibit phosphate transport by facilitating the activation of the PTH signal transduction pathway.
Open Access
Functional desensitization of the isolated beta-adrenergic receptor by the beta-adrenergic receptor kinase: potential role of an analog of the retinal protein arrestin (48-kDa protein).
(Proc Natl Acad Sci U S A, 1987-12) Benovic, JL; Kühn, H; Weyand, I; Codina, J; Caron, MG; Lefkowitz, RJ
The beta-adrenergic receptor kinase is an enzyme, possibly analogous to rhodopsin kinase, that multiply phosphorylates the beta-adrenergic receptor only when it is occupied by stimulatory agonists. Since this kinase may play an important role in mediating the process of homologous, or agonist-specific, desensitization, we investigated the functional consequences of receptor phosphorylation by the kinase and possible analogies with the mechanism of action of rhodopsin kinase. Pure hamster lung beta 2-adrenergic receptor, reconstituted in phospholipid vesicles, was assessed for its ability to mediate agonist-promoted stimulation of the GTPase activity of coreconstituted stimulatory guanine nucleotide-binding regulatory protein. When the receptor was phosphorylated by partially (approximately 350-fold) purified preparations of beta-adrenergic receptor kinase, as much as 80% inactivation of its functional activity was observed. However, the use of more highly purified enzyme preparations led to a dramatic decrease in the ability of phosphorylation to inactivate the receptor such that pure enzyme preparations (approximately 20,000-fold purified) caused only minimal (approximately 1off/- 7%) inactivation. Addition of pure retinal arrestin (48-kDa protein or S antigen), which is involved in enhancing the inactivating effect of rhodopsin phosphorylation by rhodopsin kinase, led to partial restoration of the functional effect of beta-adrenergic receptor kinase-promoted phosphorylation (41 +/- 3% inactivation). These results suggest the possibility that a protein analogous to retinal arrestin may exist in other tissues and function in concert with beta-adrenergic receptor kinase to regulate the activity of adenylate cyclase-coupled receptors.
Open Access
IFI16-STING-NF-κB signaling controls exogenous mitochondrion-induced endothelial activation.
(American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 2022-06) Li, Shu; Xu, He; Song, Mingqing; Shaw, Brian I; Li, Qi-Jing; Kirk, Allan D
Mitochondria released from injured cells activate endothelial cells (ECs), fostering inflammatory processes, including allograft rejection. The stimulator of interferon genes (STING) senses endogenous mitochondrial DNA, triggering innate immune activation via NF-κB signaling. Here, we show that exogenous mitochondria exposure induces EC STING-NF-κB activation, promoting EC/effector memory T cell adhesion, which is abrogated by NF-κB and STING inhibitors. STING activation in mitochondrion-activated ECs is independent of canonical cGMP-AMP synthetase sensing/signaling, but rather is mediated by interferon gamma-inducible factor 16 (IFI16) and can be inhibited by IFI16 inhibition. Internalized mitochondria undergo mitofusion and STING-dependent mitophagy, leading to selective sequestration of internalized mitochondria. The exposure of donor hearts to exogenous mitochondria activates murine heart ECs in vivo. Collectively, our results suggest that IFI16-STING-NF-κB signaling regulates exogenous mitochondrion-induced EC activation and mitophagy, and exogenous mitochondria foster T cell-mediated CoBRR. These data suggest a novel, donor-directed, therapeutic approach toward mitigating perioperative allograft immunogenicity.
Open Access
Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase.
(J Cell Biol, 2005-04-11) Casaletto, Jessica B; Nutt, Leta K; Wu, Qiju; Moore, Jonathan D; Etkin, Laurence D; Jackson, Peter K; Hunt, Tim; Kornbluth, Sally
Degradation of specific protein substrates by the anaphase-promoting complex/cyclosome (APC) is critical for mitotic exit. We have identified the protein Xenopus nuclear factor 7 (Xnf7) as a novel APC inhibitor able to regulate the timing of exit from mitosis. Immunodepletion of Xnf7 from Xenopus laevis egg extracts accelerated the degradation of APC substrates cyclin B1, cyclin B2, and securin upon release from cytostatic factor arrest, whereas excess Xnf7 inhibited APC activity. Interestingly, Xnf7 exhibited intrinsic ubiquitin ligase activity, and this activity was required for APC inhibition. Unlike other reported APC inhibitors, Xnf7 did not associate with Cdc20, but rather bound directly to core subunits of the APC. Furthermore, Xnf7 was required for spindle assembly checkpoint function in egg extracts. These data suggest that Xnf7 is an APC inhibitor able to link spindle status to the APC through direct association with APC core components.
Open Access
Multiple endocytic pathways of G protein-coupled receptors delineated by GIT1 sensitivity.
(Proc Natl Acad Sci U S A, 2000-02-01) Claing, A; Perry, SJ; Achiriloaie, M; Walker, JK; Albanesi, JP; Lefkowitz, RJ; Premont, RT
Recently, we identified a GTPase-activating protein for the ADP ribosylation factor family of small GTP-binding proteins that we call GIT1. This protein initially was identified as an interacting partner for the G protein-coupled receptor kinases, and its overexpression was found to affect signaling and internalization of the prototypical beta(2)-adrenergic receptor. Here, we report that GIT1 overexpression regulates internalization of numerous, but not all, G protein-coupled receptors. The specificity of the GIT1 effect is not related to the type of G protein to which a receptor couples, but rather to the endocytic route it uses. GIT1 only affects the function of G protein-coupled receptors that are internalized through the clathrin-coated pit pathway in a beta-arrestin- and dynamin-sensitive manner. Furthermore, the GIT1 effect is not limited to G protein-coupled receptors because overexpression of this protein also affects internalization of the epidermal growth factor receptor. However, constitutive agonist-independent internalization is not regulated by GIT1, because transferrin uptake is not affected by GIT1 overexpression. Thus, GIT1 is a protein involved in regulating the function of signaling receptors internalized through the clathrin pathway and can be used as a diagnostic tool for defining the endocytic pathway of a receptor.
Open Access
Phorbol esters promote alpha 1-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism.
(Proc Natl Acad Sci U S A, 1985-09) Leeb-Lundberg, LM; Cotecchia, S; Lomasney, JW; DeBernardis, JF; Lefkowitz, RJ; Caron, MG
DDT1 MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain alpha 1-adrenergic receptors (54,800 +/- 2700 sites per cell) that are coupled to stimulation of inositol phospholipid metabolism. Incubation of these cells with tumor-promoting phorbol esters, which stimulate calcium- and phospholipid-dependent protein kinase, leads to a marked attenuation of the ability of alpha 1-receptor agonists such as norepinephrine to stimulate the turnover of inositol phospholipids. This turnover was measured by determining the 32P content of phosphatidylinositol and phosphatidic acid after prelabeling of the cellular ATP pool with 32Pi. These phorbol ester-treated cells also displayed a decrease in binding affinity of cellular alpha 1 receptors for agonists with no change in antagonist affinity. By using affinity chromatography on the affinity resin Affi-Gel-A55414, the alpha 1 receptors were purified approximately equal to 300-fold from control and phorbol ester-treated 32Pi-prelabeled cells. As assessed by NaDodSO4/polyacrylamide gel electrophoresis, the Mr 80,000 alpha 1-receptor ligand-binding subunit is a phosphopeptide containing 1.2 mol of phosphate per mol of alpha 1 receptor. After phorbol ester treatment this increased to 3.6 mol of phosphate per mol of alpha 1 receptor. The effect of phorbol esters on norepinephrine-stimulated inositol phospholipid turnover and alpha 1-receptor phosphorylation showed the same rapid time course with a t1/2 less than 2 min. These results indicate that calcium- and phospholipid-dependent protein kinase may play an important role in regulating the function of receptors that are coupled to the inositol phospholipid cycle by phosphorylating and deactivating them.
Open Access
Phosphoproteomic profiling of human myocardial tissues distinguishes ischemic from non-ischemic end stage heart failure.
(PLoS One, 2014) Schechter, Matthew A; Hsieh, Michael KH; Njoroge, Linda W; Thompson, J Will; Soderblom, Erik J; Feger, Bryan J; Troupes, Constantine D; Hershberger, Kathleen A; Ilkayeva, Olga R; Nagel, Whitney L; Landinez, Gina P; Shah, Kishan M; Burns, Virginia A; Santacruz, Lucia; Hirschey, Matthew D; Foster, Matthew W; Milano, Carmelo A; Moseley, M Arthur; Piacentino, Valentino; Bowles, Dawn E
The molecular differences between ischemic (IF) and non-ischemic (NIF) heart failure are poorly defined. A better understanding of the molecular differences between these two heart failure etiologies may lead to the development of more effective heart failure therapeutics. In this study extensive proteomic and phosphoproteomic profiles of myocardial tissue from patients diagnosed with IF or NIF were assembled and compared. Proteins extracted from left ventricular sections were proteolyzed and phosphopeptides were enriched using titanium dioxide resin. Gel- and label-free nanoscale capillary liquid chromatography coupled to high resolution accuracy mass tandem mass spectrometry allowed for the quantification of 4,436 peptides (corresponding to 450 proteins) and 823 phosphopeptides (corresponding to 400 proteins) from the unenriched and phospho-enriched fractions, respectively. Protein abundance did not distinguish NIF from IF. In contrast, 37 peptides (corresponding to 26 proteins) exhibited a ≥ 2-fold alteration in phosphorylation state (p<0.05) when comparing IF and NIF. The degree of protein phosphorylation at these 37 sites was specifically dependent upon the heart failure etiology examined. Proteins exhibiting phosphorylation alterations were grouped into functional categories: transcriptional activation/RNA processing; cytoskeleton structure/function; molecular chaperones; cell adhesion/signaling; apoptosis; and energetic/metabolism. Phosphoproteomic analysis demonstrated profound post-translational differences in proteins that are involved in multiple cellular processes between different heart failure phenotypes. Understanding the roles these phosphorylation alterations play in the development of NIF and IF has the potential to generate etiology-specific heart failure therapeutics, which could be more effective than current therapeutics in addressing the growing concern of heart failure.
Open Access
Platelet-derived growth factor receptor association with Na(+)/H(+) exchanger regulatory factor potentiates receptor activity.
(Mol Cell Biol, 2000-11) Maudsley, S; Zamah, AM; Rahman, N; Blitzer, JT; Luttrell, LM; Lefkowitz, RJ; Hall, RA
Platelet-derived growth factor (PDGF) is a potent mitogen for many cell types. The PDGF receptor (PDGFR) is a receptor tyrosine kinase that mediates the mitogenic effects of PDGF by binding to and/or phosphorylating a variety of intracellular signaling proteins upon PDGF-induced receptor dimerization. We show here that the Na(+)/H(+) exchanger regulatory factor (NHERF; also known as EBP50), a protein not previously known to interact with the PDGFR, binds to the PDGFR carboxyl terminus (PDGFR-CT) with high affinity via a PDZ (PSD-95/Dlg/Z0-1 homology) domain-mediated interaction and potentiates PDGFR autophosphorylation and extracellular signal-regulated kinase (ERK) activation in cells. A point-mutated version of the PDGFR, with the terminal leucine changed to alanine (L1106A), cannot bind NHERF in vitro and is markedly impaired relative to the wild-type receptor with regard to PDGF-induced autophosphorylation and activation of ERK in cells. NHERF potentiation of PDGFR signaling depends on the capacity of NHERF to oligomerize. NHERF oligomerizes in vitro when bound with PDGFR-CT, and a truncated version of the first NHERF PDZ domain that can bind PDGFR-CT but which does not oligomerize reduces PDGFR tyrosine kinase activity when transiently overexpressed in cells. PDGFR activity in cells can also be regulated in a NHERF-dependent fashion by stimulation of the beta(2)-adrenergic receptor, a known cellular binding partner for NHERF. These findings reveal that NHERF can directly bind to the PDGFR and potentiate PDGFR activity, thus elucidating both a novel mechanism by which PDGFR activity can be regulated and a new cellular role for the PDZ domain-containing adapter protein NHERF.
Open Access
Pleiotrophin regulates the ductular reaction by controlling the migration of cells in liver progenitor niches.
(Gut, 2016-04) Michelotti, Gregory A; Tucker, Anikia; Swiderska-Syn, Marzena; Machado, Mariana Verdelho; Choi, Steve S; Kruger, Leandi; Soderblom, Erik; Thompson, J Will; Mayer-Salman, Meredith; Himburg, Heather A; Moylan, Cynthia A; Guy, Cynthia D; Garman, Katherine S; Premont, Richard T; Chute, John P; Diehl, Anna Mae
OBJECTIVE: The ductular reaction (DR) involves mobilisation of reactive-appearing duct-like cells (RDC) along canals of Hering, and myofibroblastic (MF) differentiation of hepatic stellate cells (HSC) in the space of Disse. Perivascular cells in stem cell niches produce pleiotrophin (PTN) to inactivate the PTN receptor, protein tyrosine phosphatase receptor zeta-1 (PTPRZ1), thereby augmenting phosphoprotein-dependent signalling. We hypothesised that the DR is regulated by PTN/PTPRZ1 signalling. DESIGN: PTN-GFP, PTN-knockout (KO), PTPRZ1-KO, and wild type (WT) mice were examined before and after bile duct ligation (BDL) for PTN, PTPRZ1 and the DR. RDC and HSC from WT, PTN-KO, and PTPRZ1-KO mice were also treated with PTN to determine effects on downstream signaling phosphoproteins, gene expression, growth, and migration. Liver biopsies from patients with DRs were also interrogated. RESULTS: Although quiescent HSC and RDC lines expressed PTN and PTPRZ1 mRNAs, neither PTN nor PTPRZ1 protein was demonstrated in healthy liver. BDL induced PTN in MF-HSC and increased PTPRZ1 in MF-HSC and RDC. In WT mice, BDL triggered a DR characterised by periportal accumulation of collagen, RDC and MF-HSC. All aspects of this DR were increased in PTN-KO mice and suppressed in PTPRZ1-KO mice. In vitro studies revealed PTN-dependent accumulation of phosphoproteins that control cell-cell adhesion and migration, with resultant inhibition of cell migration. PTPRZ1-positive cells were prominent in the DRs of patients with ductal plate defects and adult cholestatic diseases. CONCLUSIONS: PTN, and its receptor, PTPRZ1, regulate the DR to liver injury by controlling the migration of resident cells in adult liver progenitor niches.
Open Access
Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43.
(The Journal of cell biology, 1989-02) Skene, JH; Virág, I
Growth cones, the motile apparatus at the ends of elongating axons, are sites of extensive and dynamic membrane-cytoskeletal interaction and insertion of new membrane into the growing axon. One of the most abundant proteins in growth cone membranes is a protein designated GAP-43, whose synthesis increases dramatically in most neurons during periods of axon development or regeneration. We have begun to explore the role of GAP-43 in growth cone membrane functions by asking how the protein interacts with those membranes. Membrane-washing experiments indicate that mature GAP-43 is tightly bound to growth cone membranes, and partitioning of Triton X-114-solubilized GAP-43 between detergent-enriched and detergent-depleted phases indicates considerable hydrophobicity. The hydrophobic behavior of the protein is modulated by divalent cations, particularly zinc and calcium. In vivo labeling of GAP-43 in neonatal rat brain with [35S]methionine shows that GAP-43 is initially synthesized as a soluble protein that becomes attached to membranes posttranslationally. In tissue culture, both rat cerebral cortex cells and neuron-like PC12 cells actively incorporate [3H]palmitic acid into GAP-43. Isolated growth cones detached from their cell bodies also incorporate labeled fatty acid into GAP-43, suggesting active turnover of the fatty acid moieties on the mature protein. Hydrolysis of ester-like bonds with neutral hydroxylamine removes the bound fatty acid and exposes new thiol groups on GAP-43, suggesting that fatty acid is attached to the protein's only two cysteine residues, located in a short hydrophobic domain at the amino terminus. Modulation of the protein's hydrophobic behavior by divalent cations suggests that other domains, containing large numbers of negatively charged residues, might also contribute to GAP-43-membrane interactions. Our observations suggest a dynamic and reversible interaction of GAP-43 with growth cone membranes.
Open Access
Role of NHERF and scaffolding proteins in proximal tubule transport.
(Urological research, 2010-08) Cunningham, Rochelle; Biswas, Rajatsubhra; Steplock, Deborah; Shenolikar, Shirish; Weinman, Edward
Eukaryotic cells coordinate specific responses to hormones and growth factors by spatial and temporal organization of "signaling components." Through the formation of multiprotein complexes, cells are able to generate "signaling components" that transduce hormone signals through proteins, such as PSD-95/Dlg/ZO-1(PDZ)-containing proteins that associate by stable and dynamic interactions. The PDZ homology domain is a common protein interaction domain in eukaryotes and with greater than 500 PDZ domains identified, it is the most abundant protein interaction domain in eukaryotic cells. The NHERF (sodium hydrogen exchanger regulatory factor) proteins are PDZ domain-containing proteins that play an important role in maintaining and regulating cell function. NHERF-1 was initially identified as a brush border membrane-associated phosphoprotein essential for the cAMP/PKA-induced inhibition of the sodium hydrogen exchanger isoform 3 (NHE3). Mouse, rabbit and human renal proximal tubules also express NHERF-2 (E3KARP), a structurally related protein, which in model cell systems also binds NHE3 and mediates its inhibition by cAMP. PDZK1 (NHERF-3) and IKEPP (NHERF-4) were later identified and found to have similar homology domains, leading to their recent reclassification. Although studies have revealed similar binding partners and overlapping functions for the NHERF proteins, it is clear that there is a significant amount of specificity between them. This review focuses primarily on NHERF-1, as the prototypical PDZ protein and will give a brief summary of its role in phosphate transport and the development of some forms of nephrolithiasis.
Open Access
Tetanus toxoid and CCL3 improve dendritic cell vaccines in mice and glioblastoma patients.
(Nature, 2015-03-19) Mitchell, Duane A; Batich, Kristen A; Gunn, Michael D; Huang, Min-Nung; Sanchez-Perez, Luis; Nair, Smita K; Congdon, Kendra L; Reap, Elizabeth A; Archer, Gary E; Desjardins, Annick; Friedman, Allan H; Friedman, Henry S; Herndon, James E; Coan, April; McLendon, Roger E; Reardon, David A; Vredenburgh, James J; Bigner, Darell D; Sampson, John H
After stimulation, dendritic cells (DCs) mature and migrate to draining lymph nodes to induce immune responses. As such, autologous DCs generated ex vivo have been pulsed with tumour antigens and injected back into patients as immunotherapy. While DC vaccines have shown limited promise in the treatment of patients with advanced cancers including glioblastoma, the factors dictating DC vaccine efficacy remain poorly understood. Here we show that pre-conditioning the vaccine site with a potent recall antigen such as tetanus/diphtheria (Td) toxoid can significantly improve the lymph node homing and efficacy of tumour-antigen-specific DCs. To assess the effect of vaccine site pre-conditioning in humans, we randomized patients with glioblastoma to pre-conditioning with either mature DCs or Td unilaterally before bilateral vaccination with DCs pulsed with Cytomegalovirus phosphoprotein 65 (pp65) RNA. We and other laboratories have shown that pp65 is expressed in more than 90% of glioblastoma specimens but not in surrounding normal brain, providing an unparalleled opportunity to subvert this viral protein as a tumour-specific target. Patients given Td had enhanced DC migration bilaterally and significantly improved survival. In mice, Td pre-conditioning also enhanced bilateral DC migration and suppressed tumour growth in a manner dependent on the chemokine CCL3. Our clinical studies and corroborating investigations in mice suggest that pre-conditioning with a potent recall antigen may represent a viable strategy to improve anti-tumour immunotherapy.