Regulation of Platelet Survival by Protein Phosphatase 1 Gamma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2026-2026
Author(s):  
Masahiro Yanagisawa ◽  
Han Hyojeong ◽  
Francisca C. Gushiken ◽  
K. Vinod Vijayan
Keyword(s):  
Cell Survival ◽  
Protein Phosphatase ◽  
Conflicts Of Interest ◽  
Serine Phosphorylation ◽  
Protein Phosphatase 1 ◽  
Binding Motif ◽  
Caspase 9 ◽  
Platelet Apoptosis ◽  
Platelet Survival ◽  
Apoptotic Protein

Abstract Abstract 2026 Platelets are key players in hemostasis and their senescence is intrinsically associated with the activation of apoptotic pathways that shows similarities to the apoptosis of nucleated cells. Anti-apoptotic protein Bcl-xl restrains the pro-apoptotic Bak activity and maintains platelet survival in circulation. In nucleated cells, serine phosphorylation of a pro-apoptotic protein Bad can also promote cell survival. Serine phosphorylation of Bad is regulated by the action of serine/threonine (Ser/Thr) protein kinases and Ser/Thr protein phosphatases. Although alterations in the activities of either enzyme can change the rate of apoptosis, whether Ser/Thr phosphatases participate in regulating platelet apoptosis is unknown. In this study, we report that the mice lacking the catalytic subunit of protein phosphatase 1 gamma (PP1cγ) exhibit a moderate but significant increase in the number of circulating platelets [596 × 103/μL ± 17.9 in WT compared with 694 × 103/μL ± 19.8 in PP1cγ–/– mice; P = .0003]. Examination of the bone marrow from the PP1cγ–/– mice revealed a non-significant increase in the number of morphologically matured megakaryocytes. A trend towards decreased plasma thrombopoietin levels were also noticed in PP1cγ–/– mice. These observations suggest that an increased megakaryopoiesis/thrombopoiesis may not fully account for the increased platelet numbers in PP1cγ–/– mice. In vivo platelet survival studies revealed that the loss of PP1cγ modestly increased platelet half life in circulation (t1/2 ∼69 hours in WT compared to ∼78 hours in PP1cγ–/– mice). PP1cγ–/– platelets had decreased mean platelet volume, suggesting the PP1cγ–/– mice may harbor a greater proportion of older circulating platelets. These studies are consistent with the delayed clearance of platelets from PP1cγ–/– mice. Pro-apoptotic Bad possess PP1c binding motif and mechanistically, PP1cγ interacts with Bad protein in platelets. Phosphorylation of Bad Ser112, which promotes cell survival, was enhanced ∼50% in PP1cγ–/– platelets. Consistent with the increased BAD phosphorylation, co-immunoprecipitation studies revealed increased BAD-14-3-3 protein complexes from the PP1cγ–/– platelets. It is reported that in the phosphorylated state, BAD can interact with 14-3-3 and is sequestered in the cytoplasm, thereby preventing the binding of Bad with the mitochondrial anti-apoptotic Bcl-xl. Interaction of Bad with Bcl-xl has the potential to displace the pro-apoptotic Bak from the Bcl-xl-Bak protein complex to trigger apoptosis. Finally, immunoblotting with anti-caspase 9 antibody revealed decreased caspase 9 cleavage product (∼37 kDa) in the PP1cγ–/– platelets, suggesting decreased activation of caspase 9 dependent apoptotic pathway. These data indicate that the increased platelet counts in PP1cγ–/– mice could be in part due to delayed platelet apoptosis. Loss of PP1cγ leads to the hyperphosphorylation of BAD, which via an interaction with 14-3-3, delays caspase mediated apoptosis to prolong the life span of platelets. Disclosures: No relevant conflicts of interest to declare.

scholarly journals SARP, a new alternatively spliced protein phosphatase 1 and DNA interacting protein

2007 ◽  
Vol 402 (1) ◽  
pp. 187-196 ◽  
Author(s):  
Gareth J. Browne ◽  
Margarida Fardilha ◽  
Senga K. Oxenham ◽  
Wenjuan Wu ◽  
Nicholas R. Helps ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Transforming Growth Factor ◽  
Binding Protein ◽  
Transforming Growth Factor Β ◽  
Ankyrin Repeat ◽  
Protein Phosphatase 1 ◽  
Binding Motif ◽  
Alternative Mrna Splicing

PP1 (protein phosphatase 1) is a ubiquitously expressed serine/threonine-specific protein phosphatase whose activity towards different substrates appears to be mediated via binding to specific proteins that play critical regulatory and targeting roles. In the present paper we report the cloning and characterization of a new protein, termed SARP (several ankyrin repeat protein), which is shown to interact with all isoforms of PP1 by a variety of techniques. A region encompassing a consensus PP1-binding motif in SARP (K354VHF357) modulates endogenous SARP–PP1 activity in mammalian cells. This SARP–PP1 interaction motif lies partially within the first ankyrin repeat in contrast with other proteins [53BP2 (p53 binding protein 2), MYPT1/M110/MBS (myosin binding protein of PP1) and TIMAP (transforming growth factor β inhibited, membrane-associated protein)], where a PP1-binding motif precedes the ankyrin repeats. Alternative mRNA splicing produces several isoforms of SARP from a single human gene at locus 11q14. SARP1 and/or SARP2 (92–95 kDa) are ubiquitously expressed in all tissues with high levels in testis and sperm, where they are shown to interact with both PP1γ1 and PP1γ2. SARP3 (65 kDa) is most abundant in brain where SARP isoforms interact with both PP1α and PP1γ1. SARP is highly abundant in the nucleus of mammalian cells, consistent with the putative nuclear localization signal at the N-terminus. The presence of a leucine zipper near the C-terminus of SARP1 and SARP2, and the binding of mammalian DNA to SARP2, suggests that SARP1 and SARP2 may be transcription factors or DNA-associated proteins that modulate gene expression.

scholarly journals An eIF2α-binding motif in protein phosphatase 1 subunit GADD34 and its viral orthologs is required to promote dephosphorylation of eIF2α

2015 ◽  
Vol 112 (27) ◽  
pp. E3466-E3475 ◽  
Author(s):  
Margarito Rojas ◽  
Gabriel Vasconcelos ◽  
Thomas E. Dever
Keyword(s):  
Protein Phosphatase ◽  
African Swine Fever Virus ◽  
Consensus Sequence ◽  
Point Mutations ◽  
Translation Initiation Factor ◽  
Protein Phosphatase 1 ◽  
Initiation Factor ◽  
Protective Mechanism ◽  
Binding Motif ◽  
Α Subunit

Transient protein synthesis inhibition, mediated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), is an important protective mechanism cells use during stress conditions. Following relief of the stress, the growth arrest and DNA damage-inducible protein GADD34 associates with the broadly acting serine/threonine protein phosphatase 1 (PP1) to dephosphorylate eIF2α. Whereas the PP1-binding motif on GADD34 has been defined, it remains to be determined how GADD34 directs PP1 to specifically dephosphorylate eIF2α. In this report, we map a novel eIF2α-binding motif to the C terminus of GADD34 in a region distinct from where PP1 binds to GADD34. This motif is characterized by the consensus sequence Rx[Gnl]x1–2Wxxx[Arlv]x[Dn][Rg]xRFxx[Rlvk][Ivc], where capital letters are preferred and x is any residue. Point mutations altering the eIF2α-binding motif impair the ability of GADD34 to interact with eIF2α, promote eIF2α dephosphorylation, and suppress PKR toxicity in yeast. Interestingly, this eIF2α-docking motif is conserved among viral orthologs of GADD34, and is necessary for the proteins produced by African swine fever virus, Canarypox virus, and Herpes simplex virus to promote eIF2α dephosphorylation. Taken together, these data indicate that GADD34 and its viral orthologs direct specific dephosphorylation of eIF2α by interacting with both PP1 and eIF2α through independent binding motifs.

Non-Competitive Protein Phosphatase-1 Inhibitors Prevent Sickling of SS RBCs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4038-4038
Author(s):  
Jerod Hairston ◽  
Keon Combi ◽  
Altreisha Foster ◽  
Bak Kim ◽  
Victor R. Gordeuk ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Cultured Cells ◽  
Conflicts Of Interest ◽  
Protein Phosphatase 1 ◽  
Cell Protein ◽  
Trypan Blue Exclusion ◽  
Future Design ◽  
Small Molecular Inhibitors ◽  
Hiv 1

Abstract Abstract 4038 Poster Board III-974 Protein phosphatase-1 (PP1) has been implicated in the regulation of KCC (K:Cl) transporters, which transport K+ and Cl- ions from red blood cells (RBCs) and in the setting of sickle cell disease may contribute to RBC dehydration and sickling. We have studied host cell protein phosphatase-1 (PP1) in the context of HIV-1 replication and designed novel small molecule non-competitive inhibitors of PP1 that are efficient in the inhibition of HIV-1 but not toxic for cultured cells. We analyzed the effect of our novel non-competitive PP1 inhibitors and the conventional competitive PP1 inhibitor, ocadaic acid, on the sickling of hemoglobin SS RBCs in vitro. We cultured hemoglobin SS RBCs overnight at 1% O2 in the presence of the PP1 inhibitors and then photographed the RBCs and counted the percentage of sickled RBCs. We found that the non-competitive PP1 inhibitor, 1E7-04 prevented RBC sickling by 40% at 10 mM concentration. The 1E7-04 was not toxic at 10 mM concentration for cultured CEM T cells as determined by trypan blue exclusion assay using an automatic cell counter. Our study suggests that small molecular inhibitors of PP1 might be candidates for the future design of anti-sickling drugs. Acknowledgments. This work was supported by NHLBI grant U54HL090508-02; NHLBI grant R25 HL003679-08 from the National Institute of Helath and The Office of Research on Minority Health and by U.S. Civilian Research & Development Foundation grant. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mutations of the serine phosphorylated in the protein phosphatase-1-binding motif in the skeletal muscle glycogen-targeting subunit

2000 ◽  
Vol 346 (1) ◽  
pp. 77-82 ◽  
Author(s):  
Jun LIU ◽  
Jun WU ◽  
Carey OLIVER ◽  
Shirish SHENOLIKAR ◽  
David L. BRAUTIGAN
Keyword(s):  
Skeletal Muscle ◽  
Muscle Glycogen ◽  
Protein Phosphatase ◽  
Glycogen Metabolism ◽  
Binding Activity ◽  
Hormonal Control ◽  
Protein Phosphatase 1 ◽  
Binding Motif ◽  
Unique Contribution ◽  
Skeletal Muscle Glycogen

Cellular functions of protein phosphatase-1 (PP1) are determined by regulatory subunits that contain the consensus PP1-binding motif, RVXF. This motif was first identified as the site of phosphorylation by cAMP-dependent protein kinase (PKA) in a skeletal muscle glycogen-targeting subunit (GM). We reported previously that a recombinant fusion protein of glutathione S-transferase (GST) and the N-terminal domain of GM [GST-GM-(1-240)] bound PP1 in a pull down assay, and phosphorylation by PKA prevented PP1 binding. Here we report that substitution of either Ala or Val for Ser-67 in the RVS67F motif in GST-GM-(1-240) essentially eliminated PP1 binding. This was unexpected because other glycogen-targeting subunits have a Val residue at the position corresponding to Ser-67. In contrast, a mutation of Ser-67 to Thr (S67T) in GST-GM(1-240) gave a protein that bound PP1 the same as wild type and was unaffected by PKA phosphorylation. Full length GM tagged with the epitope sequence DYKDDDDK (FLAG) expressed in COS7 cells bound PP1 that was recovered by co-immunoprecipitation, but this association was prevented by treatment of the cells with forskolin. By comparison, PP1 binding with FLAG-GM(S67T) was not disrupted by forskolin treatment. Neither FLAG-GM(S67A) nor FLAG-GM(S67V) formed stable complexes with PP1 in COS7 cells. These results emphasise the unique contribution of Ser-67 in PP1 binding to GM. The constitutive PP1-binding activity shown by GM(S67T) opens the way for studying the role of GM multisite phosphorylation in hormonal control of glycogen metabolism.

scholarly journals Mistargeting of B-Type Lamins at the End of Mitosis

2001 ◽  
Vol 153 (3) ◽  
pp. 621-626 ◽  
Author(s):  
Rikke L. Steen ◽  
Philippe Collas
Keyword(s):  
Cell Survival ◽  
Protein Phosphatase ◽  
Nuclear Lamina ◽  
Lymphoid Cells ◽  
Protein Phosphatase 1 ◽  
Rapid Synthesis ◽  
Gene Encoding ◽  
Nuclear Assembly ◽  
Anchoring Protein

We previously showed that targeting of protein phosphatase 1 (PP1) to the nuclear envelope (NE) by the A-kinase anchoring protein, AKAP149, correlates with nuclear assembly of B-type lamins in vitro. We demonstrate here that failure of AKAP149-mediated assembly of B-type lamins into the nuclear lamina at the end of mitosis is followed by apoptosis, and induces expression of the gene encoding A-type lamins in cells that normally do not express lamins A/C. In HeLa cells, inhibition of PP1 association with the NE mediated by a peptide containing the PP1-binding domain of AKAP149 results in failure of B-type lamins to assemble, and in their rapid caspase-dependent proteolysis. However, assembly of lamins A/C is not affected. Nonetheless, apoptosis follows within hours of nuclear reformation after mitosis. In lymphoid KE37 cells, which do not express lamins A/C, inhibition of B-type lamin assembly triggers rapid synthesis and nuclear assembly of both lamins A and C before apoptosis takes place. The results indicate that nuclear assembly of B-type lamins is essential for cell survival. They also suggest that mistargeting of B-type lamins at the end of mitosis elicits a tentative rescue process to assemble a nuclear lamina in lymphoid cells that normally do not express lamins A/C.

Protein phosphatase 1 – targeted in many directions

2002 ◽  
Vol 115 (2) ◽  
pp. 241-256 ◽  
Author(s):  
Patricia T. W. Cohen
Keyword(s):  
Protein Phosphatase ◽  
Protein Phosphatase 1 ◽  
Binding Motif ◽  
Cellular Functions ◽  
Regulatory Subunits ◽  
Specific Location ◽  
Hydrophobic Groove ◽  
Intracellular Signals ◽  
Small Hydrophobic ◽  
Rvxf Motif

Protein phosphatase 1 (PP1) is a major eukaryotic protein serine/threonine phosphatase that regulates an enormous variety of cellular functions through the interaction of its catalytic subunit (PP1c) with over fifty different established or putative regulatory subunits. Most of these target PP1c to specific subcellular locations and interact with a small hydrophobic groove on the surface of PP1c through a short conserved binding motif – the RVxF motif – which is often preceded by further basic residues. Weaker interactions may subsequently enhance binding and modulate PP1 activity/specificity in a variety of ways. Several putative targeting subunits do not possess an RVxF motif but nevertheless interact with the same region of PP1c. In addition, several ‘modulator’ proteins bind to PP1c but do not possess a domain targeting them to a specific location. Most are potent inhibitors of PP1c and possess at least two sites for interaction with PP1c, one of which is identical or similar to the RVxF motif.Regulation of PP1c in response to extracellular and intracellular signals occurs mostly through changes in the levels, conformation or phosphorylation status of targeting subunits. Understanding of the mode of action of PP1c complexes may facilitate development of drugs that target particular PP1c complexes and thereby modulate the phosphorylation state of a very limited subset of proteins.

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