Rhodnius prolixus and its symbiotic actinomycete: a microbiological, physiological and behavioural study

1976 ◽  
Vol 194 (1117) ◽  
pp. 501-525 ◽  
Keyword(s):  
Blood Meal ◽  
De Novo ◽  
Wild Type ◽  
Glucose Levels ◽  
Blood Sucking ◽  
Developmental Disturbance ◽  
Novel Method ◽  
Blood Meal Digestion ◽  
Lactate Levels

The rôle of the symbiont in this blood-sucking insect has been reinvestigated by a novel method: the substitution of wild-type symbionts by auxotrophic mutants produced in vitro , unable to synthesize particular B-group vitamins namely nicotinamide, thiamin, pyridoxine, riboflavin, p -aminobenzoic acid or biotin. Symbiont-free instar IV insects infected with a mutant of any one of these phenotypes moulted to instar V essentially as though infected with wild-type symbionts and did not exhibit the substantial developmental disturbance shown by symbiont-free controls. Though occasional overgrowth of auxotrophs by prototrophic revertants occurred it was not a significant problem. Continued growth of the nicotinamide, thiamin or pyridoxine auxotroph- and wild-type-infected instar V insects was comparable. With the remaining auxotroph-infected insects results varied between experiments, possibly because of disjunction of pre- and post-ecdysial changes in the insect’s pharyngeal pump. Overall, the results are interpreted as disproving the hypothesis that the de novo synthesis of B-vitamins by the symbiont is the sine qua non of the relationship. This contrasts with the conclusions of previous workers. It is suggested that these differences might be accounted for by the more precise experimental framework adopted here. Variations on the ‘traditional’ vitamin hypothesis are suggested: these involve the ‘conversion of metabolites’ rather than the de novo synthesis of vitamins and may help reconcile conflicting results. The symbiont showed unexpected carbon source utilization in vitro and from this it is inferred that erythrocyte-mediated glycolysis within the insect’s blood-meal continues, thus converting to lactate glucose present in the insect. As blood-meal digestion progresses glycolysis would cease, leading to a possible increase in glucose levels within the gut. Cyclical changes in glucose/lactate levels might influence the development of Trypanosoma cruzi the aetiological agent of Chagas’ disease, which undergoes part of its life cycle in the triatomid gut. Peak populations of about 10 8 symbionts per instar IV insects were found, corresponding to about 5 % of the dry increase of the instar. Symbionts survived well in the insect’s faeces and this, in conjunction with the demonstrated coprophilic behaviour of the insect, which would lead to a high probability of contact with faeces, may be of critical importance in the establishment of the symbiotic relationship.

scholarly journals Glutathione Synthesis Contributes to Virulence of Streptococcus agalactiae in a Murine Model of Sepsis

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Elizabeth A. Walker ◽  
Gary C. Port ◽  
Michael G. Caparon ◽  
Blythe E. Janowiak
Keyword(s):  
Streptococcus Agalactiae ◽  
De Novo ◽  
Single Gene ◽  
Neonatal Meningitis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Glutathione Synthesis ◽  
Content Type ◽  
Resistance Pathway

ABSTRACT Streptococcus agalactiae, a leading cause of sepsis and meningitis in neonates, utilizes multiple virulence factors to survive and thrive within the human host during an infection. Unique among the pathogenic streptococci, S. agalactiae uses a bifunctional enzyme encoded by a single gene (gshAB) to synthesize glutathione (GSH), a major antioxidant in most aerobic organisms. Since S. agalactiae can also import GSH, similar to all other pathogenic streptococcal species, the contribution of GSH synthesis to the pathogenesis of S. agalactiae disease is not known. In the present study, gshAB deletion mutants were generated in strains representing three of the most prevalent clinical serotypes of S. agalactiae and were compared against isogenic wild-type and gshAB knock-in strains. When cultured in vitro in a chemically defined medium under nonstress conditions, each mutant and its corresponding wild type had comparable growth rates, generation times, and growth yields. However, gshAB deletion mutants were found to be more sensitive than wild-type or gshAB knock-in strains to killing and growth inhibition by several different reactive oxygen species. Furthermore, deletion of gshAB in S. agalactiae strain COH1 significantly attenuated virulence compared to the wild-type or gshAB knock-in strains in a mouse model of sepsis. Taken together, these data establish that GSH is a virulence factor important for resistance to oxidative stress and that de novo GSH synthesis plays a crucial role in S. agalactiae pathogenesis and further suggest that the inhibition of GSH synthesis may provide an opportunity for the development of novel therapies targeting S. agalactiae disease. IMPORTANCE Approximately 10 to 30% of women are naturally and asymptomatically colonized by Streptococcus agalactiae. However, transmission of S. agalactiae from mother to newborn during vaginal birth is a leading cause of neonatal meningitis. Although colonized mothers who are at risk for transmission to the newborn are treated with antibiotics prior to delivery, S. agalactiae is becoming increasingly resistant to current antibiotic therapies, and new treatments are needed. This research reveals a critical stress resistance pathway, glutathione synthesis, that is utilized by S. agalactiae and contributes to its pathogenesis. Understanding the role of this unique bifunctional glutathione synthesis enzyme in S. agalactiae during sepsis may help elucidate why S. agalactiae produces such an abundance of glutathione compared to other bacteria.

scholarly journals De novo disease-associated mutations in KIF1A dominant negatively inhibit axonal transport of synaptic vesicle precursors

2021 ◽  
Author(s):  
Yuzu Anazawa ◽  
Tomoki Kita ◽  
Kumiko Hayashi ◽  
Shinsuke Niwa
Keyword(s):  
Synaptic Vesicle ◽  
Axonal Transport ◽  
De Novo ◽  
Molecular Motor ◽  
Model Systems ◽  
In Vitro Assays ◽  
In Vivo Model ◽  
Wild Type

KIF1A is a kinesin superfamily molecular motor that transports synaptic vesicle precursors in axons. Mutations in Kif1a lead to a group of neuronal diseases called KIF1A-associated neuronal disorder (KAND). KIF1A forms a homodimer and KAND mutations are mostly de novo and autosomal dominant; however, it is not known whether the function of wild-type KIF1A is inhibited by disease-associated KIF1A. No reliable in vivo model systems to analyze the molecular and cellular biology of KAND have been developed; therefore, here, we established Caenorhabditis elegans models for KAND using CRISPR/cas9 technology and analyzed defects in axonal transport. In the C. elegans models, heterozygotes and homozygotes exhibited reduced axonal transport phenotypes. In addition, we developed in vitro assays to analyze the motility of single heterodimers composed of wild-type KIF1A and disease-associated KIF1A. Disease-associated KIF1A significantly inhibited the motility of wild-type KIF1A when heterodimers were formed. These data indicate the molecular mechanism underlying the dominant nature of de novo KAND mutations.

scholarly journals Assessing the blood-host plasticity and dispersal rate of the malaria vector Anopheles coluzzii

2018 ◽  
Author(s):  
James Orsborne ◽  
Luis Furuya-Kanamori ◽  
Claire L. Jeffries ◽  
Mojca Kristan ◽  
Abdul Rahim Mohammed ◽  
...  
Keyword(s):  
Blood Meal ◽  
Disease Transmission ◽  
Blood Feeding ◽  
Host Choice ◽  
Anopheles Coluzzii ◽  
Dispersal Rate ◽  
Blood Digestion ◽  
Novel Method ◽  
Blood Meal Digestion ◽  
Blood Meals

AbstractDifficulties with observing the dispersal of insect vectors in the field have hampered understanding of several aspects of their behaviour linked to disease transmission. Here, a novel method based on detection of blood-meal sources is introduced to inform two critical and understudied mosquito behaviours: plasticity in the malaria vector’s blood-host choice and vector dispersal. Strategically located collections of Anopheles coluzzii from a malaria-endemic village of southern Ghana showed statistically significant variation in host species composition of mosquito blood-meals. Trialling a new sampling approach gave the first estimates for the remarkably local spatial scale across which host choice is plastic. Using quantitative PCR, the blood-meal digestion was then quantified for field-caught mosquitoes and calibrated according to timed blood digestion in colony mosquitoes. We demonstrate how this new ‘molecular Sella score’ approach can be used to estimate the dispersal rate of blood-feeding vectors caught in the field.

scholarly journals Involvement of potD in Streptococcus pneumoniae Polyamine Transport and Pathogenesis

2006 ◽  
Vol 74 (1) ◽  
pp. 352-361 ◽  
Author(s):  
D. Ware ◽  
Y. Jiang ◽  
W. Lin ◽  
E. Swiatlo
Keyword(s):  
Streptococcus Pneumoniae ◽  
De Novo ◽  
Optimal Growth ◽  
Wild Type ◽  
Polyamine Biosynthesis ◽  
Transport Pathways ◽  
Rich Media ◽  
Polyamine Transport ◽  
Inoculation Route

ABSTRACT Polyamines such as putrescine, spermidine, and cadaverine are small, polycationic molecules that are required for optimal growth in all cells. The intracellular concentrations of these molecules are maintained by de novo synthesis and transport pathways. The human pathogen Streptococcus pneumoniae possesses a putative polyamine transporter (pot) operon that consists of the four pot-specific genes potABCD. The studies presented here examined the involvement of potD in polyamine transport and in pneumococcal pathogenesis. A potD-deficient mutant was created in the mouse-virulent serotype 3 strain WU2 by insertion duplication mutagenesis. The growth of the WU2ΔpotD mutant was identical to that of the wild-type strain WU2 in vitro in rich media. However, WU2ΔpotD possessed severely delayed growth compared to wild-type WU2 in the presence of the polyamine biosynthesis inhibitors DFMO (α-dimethyl-fluroornitithine) and MGBG [methylgloxal-bis (guanyl hydrazone)]. The mutant strain also showed a significant attenuation in virulence within murine models of systemic and pulmonary infection regardless of the inoculation route or location. These data suggest that potD is involved in pneumococcal polyamine transport and is important for pathogenesis within various infection models.

Modeling MLL-PTD Related Myelodysplastic Syndromes in Mouse.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2811-2811
Author(s):  
Xiaomei Yan ◽  
Yue Zhang ◽  
Goro Sashida ◽  
Aili Chen ◽  
Xinghui Zhao ◽  
...  
Keyword(s):  
De Novo ◽  
Conflicts Of Interest ◽  
Gene Dosage ◽  
Fetal Liver ◽  
Loss Of Function ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
De Novo Aml

Abstract Abstract 2811 MLL partial tandem duplication (MLL-PTD) is found in 5–8% of human MDS, secondary acute myeloid leukemia (s-AML) and de novo AML. The molecular and clinical features of MLL-PTD+ AML are different from MLL-fusion+ AML, although they share similar worse outcomes. Mouse knock-in model of Mll-PTD has been generated to understand its underlining mechanism (Dorrance et al. JCI. 2006). Using this model, we've recently reported hematopoietic stem/progenitor cell (HSPC) phenotypes of MllPTD/WT mice. Their HSPCs showed increased apoptosis and reduced cell number, but they have a proliferative advantage over wild-type HSPCs. Furthermore, the MllPTD/WT–derived phenotypic ST-HSCs/MPPs and even GMPs have self-renewal capabilities. However, MllPTD/WT HSPCs never develop MDS or s-AML in primary or transplanted recipient mice, suggesting that additional genetic and/or epigenetic defects are necessary for transformation (Zhang et al. Blood. 2012). Recently, high frequent co-existences of both MLL-PTD and RUNX1 mutations have been reported in several MDS, s-AML and de novo AML clinical cohorts, which strongly suggest a potential cooperation for transformation between these two mutations. Our previous study has shown that MLL interacts with and stabilizes RUNX1 (Huang et al. Blood. 2011). Thus, we hypothesize that reducing RUNX1 dosage may facilitate the MLL-PTD mediated transformation toward MDS and/or s-AML. We first generated the mice containing one allele of Mll-PTD in a Runx1+/− background and assessed HSPCs of MllPTD/wt/Runx1+/− double heterozygous (DH) mice. The DH newborns are runty; they frequently die in early postnatal stage and barely survive to adulthood, compared to the normal life span of wild type (WT) or single heterozygous (Mllwt/wt/Runx1+/− and MllPTD/wt/Runx1+/+) mice. We studied DH embryos fetal liver hematopoiesis and found reduced LSK and LSK/SLAM+ cells, partly because of increased apoptosis. Enhanced proliferation was found in DH fetal liver cells (FLCs) in vitro CFU replating assays over WT and MllPTD/wt/Runx1+/+ controls. DH FLCs also showed dominant expansion in both serial competitive and serial non-competitive BMT assays compared to WT controls. The DH derived phenotypic ST-HSCs/MPPs and GMPs also have enhanced self-renewal capabilities, rescuing hematopoiesis by giving rise to long-term repopulating cells in recipient mice better than cells derived from MllPTD/wt/Runx1+/+ mice. However, DH HSPCs didn't develop MDS or s-AML in primary or in serial BMT recipient mice. We further generated MllPTD/wt/Runx1Δ/Δ mice using Mx1-Cre mediated deletion. These mice showed thrombocytopenia 1 month after pI-pC injection, and developed pancytopenia 2–4 months later. All these MllPTD/wt/Runx1Δ/Δ mice died of MDS induced complications within 7–8 months, and tri-lineages dysplasias (TLD) were found in bone marrow aspirate. However, there are no spontaneous s-AML found in MllPTD/wt/Runx1Δ/Δ mice, which suggests that RUNX1 mutants found in MLL-PTD+ patients may not be simply loss-of-function mutations and present gain-of-function activities which cooperate with MLL-PTD in human diseases onsets. In conclusion, our study demonstrates that: 1) RUNX1 gene dosage reverse-correlates with HSPCs self-renewal activity; 2) Runx1 complete deletion causes MDS in Mll-PTD background. Future studies are needed to fully understand the collaboration between MLL-PTD and RUNX1 mutations for MDS development and leukemic transformation, which should facilitate improved therapies and patient outcomes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro.

1993 ◽  
Vol 13 (4) ◽  
pp. 2546-2553 ◽  
Author(s):  
J Posada ◽  
N Yew ◽  
N G Ahn ◽  
G F Vande Woude ◽  
J A Cooper
Keyword(s):  
Fusion Protein ◽  
Map Kinase ◽  
Xenopus Oocytes ◽  
De Novo ◽  
Rabbit Muscle ◽  
Wild Type ◽  
Kinase Activation ◽  
Mitogen Activated Protein ◽  
Map Kinase Kinase

Several protein kinases, including Mos, maturation-promoting factor (MPF), mitogen-activated protein (MAP) kinase, and MAP kinase kinase (MAPKK), are activated when Xenopus oocytes enter meiosis. De novo synthesis of the Mos protein is required for progesterone-induced meiotic maturation. Recently, bacterially synthesized maltose-binding protein (MBP)-Mos fusion protein was shown to be sufficient to initiate meiosis I and MPF activation in fully grown oocytes in the absence of protein synthesis. Here we show that MAP kinase is rapidly phosphorylated and activated following injection of wild-type, but not kinase-inactive mutant, MBP-Mos into fully grown oocytes. MAP kinase activation by MBP-Mos occurs within 20 min, much more rapidly than in progesterone-treated oocytes. The MBP-Mos fusion protein also activates MPF, but MPF activation does not occur until approximately 2 h after injection. Extracts from oocytes injected with wild-type but not kinase-inactive MBP-Mos contain an activity that can phosphorylate MAP kinase, suggesting that Mos directly or indirectly activates a MAPKK. Furthermore, activated MBP-Mos fusion protein is able to phosphorylate and activate a purified, phosphatase-treated, rabbit muscle MAPKK in vitro. Thus, in oocytes, Mos is an upstream activator of MAP kinase which may function through direct phosphorylation of MAPKK.

scholarly journals Role of Plasmodium berghei ookinete surface and oocyst capsule protein, a novel oocyst capsule-associated protein, in ookinete motility

2021 ◽  
Author(s):  
Kazuhiko Nakayama ◽  
Yuta Kimura ◽  
Yu Kitahara ◽  
Akira Soga ◽  
Asako Haraguchi ◽  
...  
Keyword(s):  
Blood Meal ◽  
Plasmodium Berghei ◽  
Protein A ◽  
Gliding Motility ◽  
Wild Type ◽  
Oocyst Wall ◽  
Infected Blood ◽  
Capsule Formation

Abstract Background: Plasmodium sp., which causes malaria, must first develop in mosquitoes before being transmitted. Upon ingesting infected blood, gametes form in the mosquito lumen, followed by fertilization and differentiation of the resulting zygotes into motile ookinetes. Within 24 hours of blood ingestion, these ookinetes traverse mosquito epithelial cells and lodge below the midgut basal lamina, where they differentiate into sessile oocysts that are protected by a capsule. Methods: We identified an ookinete surface and oocyst capsule protein (OSCP) that is involved in ookinete motility as well as oocyst capsule formation. Results: We found that knockout of OSCP in parasite decreases ookinete gliding motility and gradually reduces the number of oocysts. On day 15 after blood ingestion, the oocyst wall was significantly thinner. Moreover, adding anti-OSCP antibodies decreased the gliding speed of wild type ookinetes in vitro. Adding anti-OSCP antibodies to an infected blood meal also resulted in decreased oocyst formation. Conclusion: These findings may be useful for the development of transmission-blocking tools for malaria.

Abstract 1216: The Cardioactive Peptide Apelin Increases Adipocyte Glucose Uptake and is Necessary for the Maintenance of Systemic Insulin Sensitivity

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Patrick Yue ◽  
Tomoko Asagami ◽  
Ramendra K Kundu ◽  
Yin-Gail Yee ◽  
Alexander J Glassford ◽  
...  
Keyword(s):  
Insulin Sensitivity ◽  
Glucose Tolerance ◽  
Glucose Uptake ◽  
Embryonic Stem ◽  
Peptide Hormone ◽  
Wild Type ◽  
Glucose Levels ◽  
Glucose Tolerance Tests

Background : Apelin, a peptide hormone with unique cardioactive properties, is also an adipokine, secreted by adipocytes in response to insulin. However, the overall effect of apelin on insulin sensitivity remains largely uncharacterized. Methods : For in vitro experiments, 3T3L1 cells were differentiated into adipocytes over 8 days, with apelin (1 microM) added daily to the media. Cells were then treated with insulin (100 nM; n = 5) for 30 minutes and incubated with 2-[ 3 H]-deoxyglucose. Glucose incorporation was then measured by scintillation counting. For in vivo experiments (n = 4 all studies), apelin-deficient (KO) mice were created by homologous recombination in embryonic stem cells. At age 7 weeks, insulin and glucose tolerance tests, as well as an enzyme immunosorbent assay for insulin, were performed after a 6-hour fast. The mice were then scanned by computed tomography using a GE eXplore RS MicroCT system, and visceral adipose content was determined with MicroView software. Upon sacrifice 1 week later, visceral adipocytes were isolated via collagenase digestion, exposed to insulin, and assessed for glucose uptake as above. Results : Because apelin is upregulated by insulin in adipocytes, we measured glucose uptake in differentiated 3T3L1 cells chronically dosed with apelin. Though no differences were observed in basal uptake, insulin-induced uptake was increased versus control (p < 0.05). To further investigate the role of apelin in vivo , we assessed for insulin resistance in apelin KO mice. At 8 weeks of age, apelin KOs were heavier than age-matched wild type controls (25 vs. 22 g; p < 0.05). Though fasting glucose levels were not significantly different between groups, insulin levels were increased in the KOs (895 vs. 477 pg/microL; p < 0.05). In addition, both insulin and glucose tolerance tests were significantly abnormal in the KOs compared to wild type. Moreover, visceral fat volume was greater in the KOs (274 vs. 248 mm 3 /g body weight; p < 0.05). Finally, insulin-stimulated uptake was reduced (p < 0.05). Conclusions : Apelin is necessary for the proper maintenance of glucose homeostasis. Furthermore, apelin potentiates insulin-induced glucose uptake in adipocytes, suggesting a possible mechanism for its insulin sensitizing effects.

scholarly journals Regulation of Raf-1 and Raf-1 mutants by Ras-dependent and Ras-independent mechanisms in vitro.

1995 ◽  
Vol 15 (8) ◽  
pp. 4125-4135 ◽  
Author(s):  
P Dent ◽  
D B Reardon ◽  
D K Morrison ◽  
T W Sturgill
Keyword(s):  
Protein Kinase ◽  
Plasma Membranes ◽  
De Novo ◽  
Serine Phosphorylation ◽  
Mitogen Activated Protein Kinase ◽  
Significant Activity ◽  
Dependent Manner ◽  
Wild Type ◽  
Independent Manner

The serine/threonine kinase Raf-1 functions downstream from Ras to activate mitogen-activated protein kinase kinase, but the mechanisms of Raf-1 activation are incompletely understood. To dissect these mechanisms, wild-type and mutant Raf-1 proteins were studied in an in vitro system with purified plasma membranes from v-Ras- and v-Src-transformed cells (transformed membranes). Wild-type (His)6- and FLAG-Raf-1 were activated in a Ras- and ATP-dependent manner by transformed membranes; however, Raf-1 proteins that are kinase defective (K375M), that lack an in vivo site(s) of regulatory tyrosine (YY340/341FF) or constitutive serine (S621A) phosphorylation, that do not bind Ras (R89L), or that lack an intact zinc finger (CC165/168SS) were not. Raf-1 proteins lacking putative regulatory sites for an unidentified kinase (S259A) or protein kinase C (S499A) were activated but with apparently reduced efficiency. The kinase(s) responsible for activation by Ras or Src may reside in the plasma membrane, since GTP loading of plasma membranes from quiescent NIH 3T3 cells (parental membranes) induced de novo capacity to activate Raf-1. Wild-type Raf-1, possessing only basal activity, was not activated by parental membranes in the absence of GTP loading. In contrast, Raf-1 Y340D, possessing significant activity, was, surprisingly, stimulated by parental membranes in a Ras-independent manner. The results suggest that activation of Raf-1 by phosphorylation may be permissive for further modulation by another membrane factor, such as a lipid. A factor(s) extracted with methanol-chloroform from transformed membranes or membranes from Sf9 cells coexpressing Ras and SrcY527F significantly enhanced the activity of Raf-1 Y340D or active Raf-1 but not that of inactive Raf-1. Our findings suggest a model for activation of Raf-1, wherein (i) Raf-1 associates with Ras-GTP, (ii) Raf-1 is activated by tyrosine and/or serine phosphorylation, and (iii) Raf-1 activity is further increased by a membrane cofactor.

Functional Regulation of the GPIb-IX-V Complex by 14-3-3ζ: Role of Protein Kinase A Phosphorylation of Multiple GPIbα Cytoplasmic Residues.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3654-3654
Author(s):  
Adam D. Munday ◽  
Christopher M. Diangco ◽  
Jose A. Lopez
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Platelet Function ◽  
De Novo ◽  
Cytoplasmic Domain ◽  
Wild Type ◽  
Functional Regulation ◽  
Kinase A

Abstract Platelet adhesion to the site of vascular injury is essential to prevent blood loss. The initial step of adhesion is mediated by the glycoprotein (GP) Ib-IX-V complex on the platelet surface, binding von Willebrand factor (VWF) on the exposed subendothelium. This interaction is transitory, resulting in platelet rolling, and elicits “inside-out” activation of the integrin αIIβ3, thus instigating stable arrest of platelets on fibrinogen and/or VWF and their subsequent spreading and aggregation. The GPIb-IX-V complex consists of 4 polypeptides: GPIbα disulfide linked to GPIbβ, GPIX, and GPV. While recent effort has focused on elucidation of GPIb-IX-V-generated signals, much remains to be learned. Each component of the complex is a type I transmembrane protein, possessing a C-terminal cytoplasmic tail. Of these, GPIbα’s is the longest at 96 amino acids, and associates with both signaling molecules (PI 3-kinase and Src kinase) and structural proteins (filamin A and 14-3-3ζ). Yet, the GPIbα cytoplasmic sequence lacks domains used by other receptors to recruit signaling molecules. Its only tyrosine residue, at amino acid 605, is not within a known consensus sequence for phosphorylation. However, 10 serine and 8 threonine residues are contained within the cytoplasmic domain. Of these, S587, S590, and S609 are known to be stably phosphorylated in resting platelets and to facilitate 14-3-3ζ binding. S609 does not reside in a consensus motif for phosphorylation, whereas S590 and S587 are within consensus motifs for Casein kinase I and the cAMP-dependent protein kinase A (PKA), respectively. Two other residues, T547 and S566, also reside within consensus sites for PKA. PKA has previously been demonstrated to phosphorylate S166 of GPIbβ and thereby inhibit platelet function. We hypothesized that phosphorylation of GPIbα by PKA also regulates platelet function and 14-3-3ζ binding. To test this, we produced a recombinant protein comprising the cytoplasmic domain of GPIbα (amino acids 515–610) fused to glutathione S-transferase at its N-terminus and evaluated the ability of PKA to phosphorylate the protein in vitro. Once we established that PKA could indeed phosphorylate the protein, we produced the recombinant in bacteria also expressing the PKA catalytic domain in an effort to phosphorylate the recombinant GPIbα cytoplasmic domain de novo to avoid cumbersome in vitro phosphorylations and increase the yield. Analysis using a phosphoS609 antibody demonstrated that GPIbα was phosphorylated on S609. We also examined 14-3-3 binding to wild type and mutant GPIbα expressed as part of the GPIb-IX complex in CHO cells (CHOαβIX) by evaluating which proteins were pulled down with GST-14-3-3ζ from lysates. 14-3-3ζ was able to pull down wild-type GPIbα, but only 5–10% as much of GPIbα S609A. Combined mutation of T547 and S609, each to A, completely abrogated 14-3-3ζ binding, as did combined mutation to A of T547, S566, S587, S590 and S609. Interestingly, approximately 20% residual binding was observed for GPIbα S587A/S590A and GPIbα T547A/S587A/S590A/S609A. These results indicate that PKA phosphorylates T547, S566, S587, S590 and S609 in vitro and at least S609 de novo in bacteria. They also demonstrate that 14-3-3ζ can associate with the cytoplasmic tail of GPIbα via residues T547, S566, S587, S590 and S609. This suggests a complex pattern of functional regulation of the GPIb-IX-V complex by PKA mediated through differential binding of 14-3-3ζ, involving both GPIbα and GPIbβ.

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