CIN85 Is a New Protein Phosphatase 2Ac Interacting Protein That Regulates αIIbβ3 Adhesion to Fibrinogen

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 95-95
Author(s):  
Tanvir Khatlani ◽  
Subhashree Pradhan ◽  
Vladimir Buchman ◽  
K. Vinod Vijayan
Keyword(s):  
Protein Kinases ◽  
Platelet Activation ◽  
Protein Phosphatase ◽  
Adaptor Protein ◽  
Rich Region ◽  
Interacting Protein ◽  
Integrin Αiibβ3 ◽  
293 Cells ◽  
Proline Rich Region

Abstract Abstract 95 Platelet activation at the site of injury is dependent on signal transduction events that are mediated by protein kinases and protein phosphatases. Reversible tyrosine and/or serine/threonine phosphorylation dependent assembly of effector (cytoskeletal, signaling and adaptor) proteins are critical for propagating signaling downstream of platelet receptors. Several studies have indicated a key role for protein kinases and their effectors in regulating the functions of integrin αIIbβ3. In contrast, much less is known about the contribution of serine/threonine phosphatases in integrin function, and the identities of effectors regulated by phosphatases are unknown. In this context, we have previously noticed that depletion of the catalytic subunit of protein phosphatase 2A (PP2Ac) enhanced Src activation and augmented αIIbβ3 adhesiveness to immobilized fibrinogen. Since protein-protein interactions form the foundation of cell signaling networks, we sought to identify the potential effectors of PP2Ac. We employed yeast two-hybrid interaction studies with the full length PP2Ac fused to GAL4 binding domain as bait and screened human bone marrow library. A novel interaction of PP2Ac with a protein called CIN85 was identified. Although CIN85 associates with several proteins, an interaction with PP2Ac has not been reported in any cell types. CIN85 (Cbl-interacting protein of 85 kDa) also known as Ruk or SETA is an adaptor protein with three SH3 domains, followed by a proline rich region, a serine rich region and a coiled-coil region. CIN85 participates in vesicle mediated transport and cytoskeleton remodeling. Co-immunoprecipitation (co-IP) experiments validated the interaction of the HA tagged PP2Ac with FLAG tagged CIN85 in 293 cells expressing PP2Ac-HA and CIN85-FLAG. Purified PP2Ac bound to recombinant CIN85-GST protein but not to GST protein, indicating that the in vitro interaction of PP2Ac with CIN85 was direct. Transfection and co-IP experiments with several FLAG tagged truncation mutants of CIN85 in 293 cells revealed that the interaction of PP2Ac with CIN85 was mediated by the proline rich region of CIN85. These studies established a direct interaction of PP2Ac with CIN85. Importantly, the interaction of purified PP2Ac with recombinant CIN85 decreased PP2Ac activity, suggesting that this complex has signaling consequence in vitro. We explored and showed for the first time that CIN85 is expressed in platelets. More importantly, PP2Ac co-immunoprecipitated with CIN85 in human platelets and in 293 αIIbβ3 cells suspended over BSA substrate. Interestingly, adhesion of platelets and 293 αIIbβ3 cells to immobilized fibrinogen induced dissociation of this complex. These studies suggest that the dissociation of PP2Ac-CIN85 complex following integrin stimulation enables CIN85 to propagate outside-in signals by efficiently engaging with other downstream effectors. Consistent with this notion, siRNA mediated depletion of CIN85 significantly (p<0.001) decreased adhesion of 293 αIIbβ3 cells to immobilized fibrinogen. These studies reveal that platelet activation events involve the coupling of the integrin αIIbβ3 adhesion initiated signaling with the phosphatase effector CIN85. Disclosures: No relevant conflicts of interest to declare.

Gβ1 a Component Of The Heterotrimeric G Protein Is a New Protein Phosphatase 1c Interacting Protein That Regulates Platelet Activation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3508-3508
Author(s):  
Subhashree Pradhan ◽  
Tanvir Khatlani ◽  
Satya P. Kunapuli ◽  
K. Vinod Vijayan
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
Platelet Activation ◽  
Protein Interactions ◽  
Protein Phosphatase ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Adaptor Protein ◽  
Gpcr Signaling

Abstract Platelet activation at the site of injury is tied to signal transduction events that are mediated by protein kinases and phosphatases. Reversible tyrosine, serine/threonine (Ser/Thr) phosphorylation-dependent assembly and/or disassembly of effector (cytoskeletal, signaling and adaptor) protein complexes propagate signaling downstream of G protein coupled receptors (GPCRs). Compared to kinases, the contribution of Ser/Thr phosphatases and its effectors in GPCR signaling studies is not well explored. Our previous studies had revealed that the catalytic subunit of protein phosphatase 1γ (PP1cγ) support GPCR signaling and thrombus formation. Since cell signaling networks are dependent on protein-protein interactions, we sought to identify the potential effectors of PP1cγ. We employed yeast two-hybrid interaction studies with the full length PP1cγ fused to GAL4 activating domain as bait and screened human bone marrow library. A novel interaction of PP1cγ with a protein called Gβ1 (GNB1) was identified. Gβ1 is a component of the heterotrimeric G proteins like the Gα and couple to GPCR. However, unlike Gα subunits, Gβ1 is unexplored in platelets. Co-immunoprecipitation (co-IP) studies validated PP1cγ-Gβ1 interaction in 293 cells expressing PP1cγ-HA and Gβ1-FLAG. Importantly, Gβ1 interacted with all the PP1c isoforms, suggesting that Gβ1 could target all PP1c isoforms to the GPCR complex. Purified PP1c bound to recombinant Gβ1-GST protein but not to GST protein, indicating that the in vitro interaction of PP1c with Gβ1 was direct and independent of Gα and Gγ subunits. A small molecule inhibitor of G protein βγ, gallein decreased thrombin-induced human platelet aggregation and adhesion to immobilized fibrinogen. There is a paucity of Gβ1-/- platelets because Gβ1-/- mice die within 2 days of birth due to microencephaly. siRNA mediated depletion of Gβ1 in murine megakaryocytes reduced PAR4-activating peptide induced soluble fibrinogen binding to αIIbβ3. These studies suggest a functional role for Gβ1 in GPCR signaling. PP1c co-immunoprecipitated with Gβ1 in resting platelets and agonist (thrombin and ADP) treatment under non-stirring conditions induced dissociation of PP1c from Gβ1. These studies indicate that Gβ1-PP1c complex in platelets is responsive to agonist. Furthermore, PP1c and Gβ1 associated with P2Y12 receptor in resting but not agonist activated platelets in a co-IP assay, suggesting a role for this complex in G protein signaling. Finally, agonist induced dissociation of PP1c from Gβ1 correlated with the association of PP1c with the downstream GPCR effector phospholipase C β3 (PLCβ3) with a concomitant dephosphorylation of PLCβ3 at Ser1105. Since previous studies have revealed that PLCβ3 activity is inhibited by Ser1105 phosphorylation, our observation suggest that agonist-induced association of PP1c with PLCβ3 facilitates dephosphorylation and activation of PLCβ3. These studies highlight a coupling of GPCR signaling with the phosphatase driven signal transduction during platelet activation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Branched-Chain Amino Acid Catabolism Promotes Thrombosis Risk by Enhancing Tropomodulin-3 Propionylation in Platelets

Circulation ◽  
2020 ◽  
Vol 142 (1) ◽  
pp. 49-64 ◽  
Author(s):  
Yanyan Xu ◽  
Haojie Jiang ◽  
Li Li ◽  
Fengwu Chen ◽  
Yunxia Liu ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Platelet Activation ◽  
Protein Phosphatase ◽  
Arterial Thrombosis ◽  
Chinese Hamster Ovary Cells ◽  
Platelet Activity ◽  
Integrin Αiibβ3 ◽  
Thrombosis Risk ◽  
Mass Spectrometry Identification ◽  
Branched Chain

Background: Branched-chain amino acids (BCAAs), essential nutrients including leucine, isoleucine, and valine, serve as a resource for energy production and the regulator of important nutrient and metabolic signals. Recent studies have suggested that dysfunction of BCAA catabolism is associated with the risk of cardiovascular disease. Platelets play an important role in cardiovascular disease, but the functions of BCAA catabolism in platelets remain unknown. Methods: The activity of human platelets from healthy subjects before and after ingestion of BCAAs was measured. Protein phosphatase 2Cm specifically dephosphorylates branched-chain α-keto acid dehydrogenase and thereby activates BCAA catabolism. Protein phosphatase 2Cm–deficient mice were used to elucidate the impacts of BCAA catabolism on platelet activation and thrombus formation. Results: We found that ingestion of BCAAs significantly promoted human platelet activity (n=5; P <0.001) and arterial thrombosis formation in mice (n=9; P <0.05). We also found that the valine catabolite α-ketoisovaleric acid and the ultimate oxidation product propionyl-coenzyme A showed the strongest promotion effects on platelet activation, suggesting that the valine/α-ketoisovaleric acid catabolic pathway plays a major role in BCAA-facilitated platelet activation. Protein phosphatase 2Cm deficiency significantly suppresses the activity of platelets in response to agonists (n=5; P <0.05). Our results also suggested that BCAA metabolic pathways may be involved in the integrin αIIbβ3–mediated bidirectional signaling pathway that regulates platelet activation. Mass spectrometry identification and immunoblotting revealed that BCAAs enhanced propionylation of tropomodulin-3 at K255 in platelets or Chinese hamster ovary cells expressing integrin αIIbβ3. The tropomodulin-3 K255A mutation abolished propionylation and attenuated the promotion effects of BCAAs on integrin-mediated cell spreading, suggesting that K255 propionylation of tropomodulin-3 is an important mechanism underlying integrin αIIbβ3–mediated BCAA-facilitated platelet activation and thrombosis formation. In addition, the increased levels of BCAAs and the expression of positive regulators of BCAA catabolism in platelets from patients with type 2 diabetes mellitus are significantly correlated with platelet hyperreactivity. Lowering dietary BCAA intake significantly reduced platelet activity in ob/ob mice (n=4; P <0.05). Conclusions: BCAA catabolism is an important regulator of platelet activation and is associated with arterial thrombosis risk. Targeting the BCAA catabolism pathway or lowering dietary BCAA intake may serve as a novel therapeutic strategy for metabolic syndrome–associated thrombophilia.

scholarly journals Mammalian Septins Regulate Microtubule Stability through Interaction with the Microtubule-binding Protein MAP4

2005 ◽  
Vol 16 (10) ◽  
pp. 4648-4659 ◽  
Author(s):  
Brandon E. Kremer ◽  
Timothy Haystead ◽  
Ian G. Macara
Keyword(s):  
Mammalian Cells ◽  
Spectroscopic Analysis ◽  
Molecular Function ◽  
Rich Region ◽  
Intact Cells ◽  
Pronounced Increase ◽  
Binding Partner ◽  
Microtubule Stability ◽  
Proline Rich Region

Mammalian septins constitute a family of at least 12 GTP-binding proteins that can form hetero-oligomers and that are sometimes found in association with actin or microtubule filaments. However, their functions are not understood. Using RNA interference, we found that suppression of septin expression in HeLa cells caused a pronounced increase in microtubule stability. Mass spectroscopic analysis of proteins coprecipitating with Sept6 identified the microtubule-associated protein MAP4 as a septin binding partner. A small, proline-rich region in the C-terminal half of MAP4 bound directly to a Sept 2:6:7 heterotrimer, and to the Sept2 monomer. The trimer blocked the ability of this MAP4 fragment to bind and bundle microtubules in vitro. In intact cells, MAP4 was required for the stabilization of microtubules induced by septin depletion. Moreover, septin depletion increased the number of cells with abnormal nuclei, and this effect was blocked by gene silencing of MAP4. These data identify a novel molecular function for septins in mammalian cells: the modulation of microtubule dynamics through interaction with MAP4.

scholarly journals The phosphorylation of CapZ-interacting protein (CapZIP) by stress-activated protein kinases triggers its dissociation from CapZ

2005 ◽  
Vol 389 (1) ◽  
pp. 127-135 ◽  
Author(s):  
Claire E. EYERS ◽  
Helen McNEILL ◽  
Axel KNEBEL ◽  
Nick MORRICE ◽  
Simon J. C. ARTHUR ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Osmotic Shock ◽  
Jurkat Cells ◽  
Mitogen Activated Protein Kinase ◽  
Interacting Protein ◽  
Capping Protein ◽  
Sb 203580

A protein expressed in immune cells and muscle was detected in muscle extracts as a substrate for several SAPKs (stress-activated protein kinases). It interacted specifically with the F-actin capping protein CapZ in splenocytes, and was therefore termed ‘CapZIP’ (CapZ-interacting protein). Human CapZIP was phosphorylated at Ser-179 and Ser-244 by MAPKAP-K2 (mitogen-activated protein kinase-activated protein kinase 2) or MAPKAP-K3 in vitro. Anisomycin induced the phosphorylation of CapZIP at Ser-179 in Jurkat cells, which was prevented by SB 203580, consistent with phosphorylation by MAPKAP-K2 and/or MAPKAP-K3. However, osmotic shock-induced phosphorylation of Ser-179 was unaffected by SB 203580. These and other results suggest that CapZIP is phosphorylated at Ser-179 in cells by MAPKAP-K2/MAPKAP-K3, and at least one other protein kinase. Stress-activated MAP kinase family members phosphorylated human CapZIP at many sites, including Ser-68, Ser-83, Ser-108 and Ser-216. Ser-108 became phosphorylated when Jurkat cells were exposed to osmotic shock, which was unaffected by SB 203580 and/or PD 184352, or in splenocytes from mice that do not express either SAPK3/p38γ or SAPK4/p38δ. Our results suggest that CapZIP may be phosphorylated by JNK (c-Jun N-terminal kinase), which phosphorylates CapZIP to >5 mol/mol within minutes in vitro. Osmotic shock or anisomycin triggered the dissociation of CapZIP from CapZ in Jurkat cells, suggesting that phosphorylation of CapZIP may regulate the ability of CapZ to remodel actin filament assembly in vivo.

The Acetyl-Transferase Tip60 Interacts with c-Myb and Inactivates Its Transcriptional Activity in Human Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3981-3981
Author(s):  
Huiwu Zhao ◽  
Shenghao Jin ◽  
Alan M. Gewirtz
Keyword(s):  
Histone Deacetylases ◽  
Transcriptional Activity ◽  
Hematopoietic Cells ◽  
Adaptor Protein ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Transactivation Domain ◽  
Interacting Protein ◽  
Factor C

Abstract Abstract 3981 Poster Board III-917 The acetyl-transferaseTip60 (Tat interacting protein, 60 kDa; also known as K(lysine) acetyltransferase 5:KAT5) is a co-regulator of transcription factors and is implicated in tumorigenesis. The protooncogene c-myb encodes a transcription factor, c-Myb, which is essential for normal hematopoisis and promotes hematologic malignancies. In this study, we explored potential regulatory relationships between Tip60 and c-Myb in hematopoietic cells. We first sought to detect any direct physical interactions by performing co-immunoprecitation (co-IP) assays. These revealed that Tip60 associated with c-Myb in Jurkat (T leukemia) and K562 (CML) cells. In vitro translated, epitope-tagged c-Myb and Tip60 also interacted with each other, suggesting that the Tip60, c-Myb interaction did not require an adaptor protein or co-factor. We then sought to define the interacting protein domains. Deletion analysis studies revealed that the interaction between Tip60 and c-Myb was dependent on the Tip60 MYST acetytransferase domain and transactivation domain of c-Myb. We then determined whether Tip60 could acetylate c-Myb, a post-translational event known to modulate c-Myb activity. Interestingly, an in vitro acetyltransferase assay showed that c-Myb was not a substrate of Tip60, even though Tip60 acetylated itself in the same assay. We then examined the effect of Tip60 on the ability of c-Myb to transcriptionally activate known target genes. In HEK293T cells, co-expressing Tip60 with c-Myb decreased c-Myb's ability to activate a luciferase reporter driven by the cim-1 promoter, a verified c-Myb target, by ∼60% compared to activation in the absence of Tip60. The physiologic significance of this observation was then explored. A chromatin immunoprecipiation (ChIP) assay revealed that Tip60 bound to the c-myc promoter, another known c-Myb target gene, in K562 cells. Furthermore, inactivation of endogenous c-Myb in K562 cells stably expressing an inducible c-Myb DNA binding domain reduced the occupancy of Tip60 in the c-myc promoter, suggesting that Tip60 utilizes c-Myb to bind its preferred site in the c-myc promoter. Using c-Myb, Tip60, and appropriate control siRNAs we achieved specific knockdowns of c-Myb, and Tip60 (∼80-90%, and ∼70-80% respectively compared to controls). Consistent with prior reports, c-myc expression decreased ∼60% when c-Myb was targeted, and ∼50% increased when Tip60 was targeted. A mechanistic explanation was sought to explain this finding. Tip60 is represses transcription when associated with histone deacetylases (HDAC), including HDAC1, HDAC2 and HDAC7. Co-IP of Jurkat cell lysates revealed that c-Myb is associated with HDAC1 and HDAC2. Altogether, these data suggest that Tip60 directly associates with c-Myb, and may inhibit its transcriptional activity by recruiting histone deacetylases(HDAC1 and HDAC2) to the activation complex. Finally, we compared Tip60 expression in 6 primary AML samples, with 3 normal CD34+ cell samples using QRT-PCR. Tip60 expression was significantly (∼60%) lower in the AML samples. In summary, these studies demonstrate that Tip60 modulates c-Myb transcriptional activity in human hematopoietic cells leading us to hypothesize that Tip60 is a normal regulator of c-Myb function and that dysregulated or mutated Tip60 may contribute to c-Myb driven leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

Suppression of Murine Platelet Activation by the β Isoform of the Catalytic Subunit of Protein Phosphatase 2B

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 190-190
Author(s):  
Francisca C. Gushiken ◽  
Nawaf Alrehani ◽  
Subhashree Pradhan ◽  
Lavanya Kailasam ◽  
Rolando Rumbaut ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Protein Phosphatase ◽  
Conflicts Of Interest ◽  
Fibrin Clot ◽  
Catalytic Subunit ◽  
Low Doses ◽  
Clot Retraction ◽  
Protein Phosphatase 2B ◽  
Platelet Functions

Abstract Abstract 190 Signal transduction mediated by the kinases and phosphatases are critical for platelet activation at the site of vascular injury. Compared to the kinases, a role for phosphatases in platelet activation is less understood. Our previous studies have focused on the roles of serine/threonine protein phosphatase 1 (PP1) and 2A (PP2A) in regulating integrin αIIbβ3functions. However, platelets also express protein phosphatase 2B (PP2B) and its role in platelet function is unexplored. PP2B-Aα and PP2B-Aβ constitute two ubiquitous isoforms of the PP2B catalytic subunit. Due to the general concerns regarding the specificity of the PP2B inhibitors, we have utilized mice deficient in the β isoform of the catalytic subunit of PP2B (PP2B-Aβ) to explore the role of PP2B in platelet functions. Mice lacking PP2B-Aα are short lived and are not considered in this study. Loss of PP2B-Aβ did not cause any compensatory increase in the PP2B-Aα levels in platelets. Compared to the wild type (WT) platelets, PP2B-Aβ−/− platelets displayed increased aggregation in response to low doses of protease-activated receptor 4-activating peptide (PAR4-AP), ADP, collagen and collagen related peptide (CRP). Enhanced α granule secretion in response to the low doses of PAR4-AP and CRP was noticed in PP2B-Aβ−/− platelets, relative to the WT platelets. Functions regulated by the outside-in αIIbβ3 integrin signaling like adhesion to immobilized fibrinogen and fibrin clot retraction were enhanced in the PP2B-Aβ−/− platelets. These studies indicate that PP2B-Aβ negatively regulate platelet functions in vitro. Consistent with these observations, PP2B-Aβ−/− mice exhibited a shorter tail bleeding time compared to the WT mice. In a FeCl3 induced endothelial denudation injury model, PP2B-Aβ−/− mice showed decreased time to occlusion in the carotid artery, and reduced number of emboli compared to the WT mice. These studies indicate that PP2B-Aβ suppress multiple murine platelet functions that contribute to an occlusive thrombi. Unlike a positive thrombus promoting role for the PP1cγ that was noticed in our previous study, PP2B-Aβ suppressed murine platelet activation, suggesting that different subtypes of Ser/Thr phosphatases have distinct roles in murine platelet activation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Extracellular Signal-Regulated Kinase 1 Interacts with and Phosphorylates CdGAP at an Important Regulatory Site

2005 ◽  
Vol 25 (15) ◽  
pp. 6314-6329 ◽  
Author(s):  
Joseph Tcherkezian ◽  
Eric I. Danek ◽  
Sarah Jenna ◽  
Ibtissem Triki ◽  
Nathalie Lamarche-Vane
Keyword(s):  
Bound States ◽  
Mitogen Activated Protein Kinase ◽  
Regulatory Site ◽  
Nucleotide Exchange ◽  
Rich Region ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Proline Rich Region

ABSTRACT Rho GTPases regulate multiple cellular processes affecting both cell proliferation and cytoskeletal dynamics. Their cycling between inactive GDP- and active GTP-bound states is tightly regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We have previously identified CdGAP (for Cdc42 GTPase-activating protein) as a specific GAP for Rac1 and Cdc42. CdGAP consists of an N-terminal RhoGAP domain and a C-terminal proline-rich region. In addition, CdGAP is a member of the impressively large number of mammalian RhoGAP proteins that is well conserved among both vertebrates and invertebrates. In mice, we find two predominant isoforms of CdGAP differentially expressed in specific tissues. We report here that CdGAP is highly phosphorylated in vivo on serine and threonine residues. We find that CdGAP is phosphorylated downstream of the MEK-extracellular signal-regulated kinase (ERK) pathway in response to serum or platelet-derived growth factor stimulation. Furthermore, CdGAP interacts with and is phosphorylated by ERK-1 and RSK-1 in vitro. A putative DEF (docking for ERK FXFP) domain located in the proline-rich region of CdGAP is required for efficient binding and phosphorylation by ERK1/2. We identify Thr776 as an in vivo target site of ERK1/2 and as an important regulatory site of CdGAP activity. Together, these data suggest that CdGAP is a novel substrate of ERK1/2 and mediates cross talk between the Ras/mitogen-activated protein kinase pathway and regulation of Rac1 activity.

scholarly journals Identification of Tetratricopeptide Repeat 1 as an Adaptor Protein That Interacts with Heterotrimeric G Proteins and the Small GTPase Ras

2003 ◽  
Vol 23 (11) ◽  
pp. 3847-3858 ◽  
Author(s):  
Caroline Marty ◽  
Darren D. Browning ◽  
Richard D. Ye
Keyword(s):  
G Proteins ◽  
Mammalian Cells ◽  
Active Form ◽  
Adaptor Protein ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Tetratricopeptide Repeat ◽  
Heterotrimeric G Proteins ◽  
Interacting Protein

ABSTRACT The biological functions of heterotrimeric G proteins and small GTPases are modulated by both extracellular stimuli and intracellular regulatory proteins. Using Saccharomyces cerevisiae two-hybrid screening, we identified tetratricopeptide repeat 1 (TPR1), a 292-amino-acid protein with three TPR motifs, as a Gα16-binding protein. The interaction was confirmed both in vitro and in transfected mammalian cells, where TPR1 also binds to several other Gα proteins. TPR1 was found to interact with Ha-Ras preferentially in its active form. Overexpression of TPR1 promotes accumulation of active Ras. TPR1 was found to compete with the Ras-binding domain (RBD) of Raf-1 for binding to the active Ras, suggesting that it may also compete with Ras GTPase-activating protein, thus contributing to the accumulation of GTP-bound Ras. Expression of Gα16 strongly enhances the interaction between TPR1 and Ras. Removal of the TPR1 N-terminal 112 residues abolishes potentiation by Gα16 while maintaining the interaction with Gα16 and the ability to discriminate active Ras from wild-type Ras. We have also observed that LGN, a Gαi-interacting protein with seven TPR motifs, binds Ha-Ras. Thus, TPR1 is a novel adaptor protein for Ras and selected Gα proteins that may be involved in protein-protein interaction relating to G-protein signaling.

scholarly journals A Proline-Rich Region in the Coxsackievirus 3A Protein Is Required for the Protein To Inhibit Endoplasmic Reticulum-to-Golgi Transport

2005 ◽  
Vol 79 (8) ◽  
pp. 5163-5173 ◽  
Author(s):  
Els Wessels ◽  
Daniël Duijsings ◽  
Richard A. Notebaart ◽  
Willem J. G. Melchers ◽  
Frank J. M. van Kuppeveld
Keyword(s):  
Endoplasmic Reticulum ◽  
Structural Model ◽  
Ectopic Expression ◽  
Rna Replication ◽  
Secretory Protein ◽  
Viral Rna ◽  
Rich Region ◽  
Golgi Transport ◽  
Proline Rich Region

ABSTRACT The ability of the 3A protein of coxsackievirus B (CVB) to inhibit protein secretion was investigated for this study. Here we show that the ectopic expression of CVB 3A blocked the transport of both the glycoprotein of vesicular stomatitis virus, a membrane-bound secretory marker, and the alpha-1 protease inhibitor, a luminal secretory protein, at a step between the endoplasmic reticulum (ER) and the Golgi complex. CVB 3A contains a conserved proline-rich region in its N terminus. The importance of this proline-rich region was investigated by introducing Pro-to-Ala substitutions. The mutation of Pro19 completely abolished the ability of 3A to inhibit ER-to-Golgi transport. The mutation of Pro14, Pro17, or Pro20 also impaired this ability, but to a lesser extent. The mutation of Pro18 had no effect. We also investigated the possible importance of this proline-rich region for the function of 3A in viral RNA replication. To this end, we introduced the Pro-to-Ala mutations into an infectious cDNA clone of CVB3. The transfection of cells with in vitro-transcribed RNAs of these clones gave rise to mutant viruses that replicated with wild-type characteristics. We concluded that the proline-rich region in CVB 3A is required for its ability to inhibit ER-to-Golgi transport, but not for its function in viral RNA replication. The functional relevance of the proline-rich region is discussed in light of the proposed structural model of 3A.

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