Rice LecRK5 phosphorylates a UGPase to regulate callose biosynthesis during pollen development

Abstract The temporary callose layer surrounding the tetrads of microspores is critical for male gametophyte development in flowering plants, as abnormal callose deposition can lead to microspore abortion. A sophisticated signaling network regulates callose biosynthesis but these pathways are poorly understood. In this study, we characterized a rice male-sterile mutant, oslecrk5, which showed defective callose deposition during meiosis. OsLecRK5 encodes a plasma membrane-localized lectin receptor-like kinase, which can form a dimer with itself. Moreover, normal anther development requires the K-phosphorylation site (a conserved residue at the ATP-binding site) of OsLecRK5. In vitro assay showed that OsLecRK5 phosphorylates the callose synthesis enzyme UGP1, enhancing callose biosynthesis during anther development. Together, our results demonstrate that plasma membrane-localized OsLecRK5 phosphorylates UGP1 and promotes its activity in callose biosynthesis in rice. This is the first evidence that a receptor-like kinase positively regulates callose biosynthesis.

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OsMYB80 Regulates Anther Development and Pollen Fertility by Targeting Multiple Biological Pathways

Plant and Cell Physiology ◽

10.1093/pcp/pcaa025 ◽

2020 ◽

Vol 61 (5) ◽

pp. 988-1004 ◽

Cited By ~ 3

Author(s):

Xiaoying Pan ◽

Wei Yan ◽

Zhenyi Chang ◽

Yingchao Xu ◽

Ming Luo ◽

...

Keyword(s):

Pollen Development ◽

Male Fertility ◽

Gene Networks ◽

Affinity Purification ◽

Anther Development ◽

Rna Seq ◽

Male Sterile ◽

New Information ◽

Gel Shift Assay

Abstract Pollen development is critical to the reproductive success of flowering plants, but how it is regulated is not well understood. Here, we isolated two allelic male-sterile mutants of OsMYB80 and investigated how OsMYB80 regulates male fertility in rice. OsMYB80 was barely expressed in tissues other than anthers, where it initiated the expression during meiosis, reached the peak at the tetrad-releasing stage and then quickly declined afterward. The osmyb80 mutants exhibited premature tapetum cell death, lack of Ubisch bodies, no exine and microspore degeneration. To understand how OsMYB80 regulates anther development, RNA-seq analysis was conducted to identify genes differentially regulated by OsMYB80 in rice anthers. In addition, DNA affinity purification sequencing (DAP-seq) analysis was performed to identify DNA fragments interacting with OsMYB80 in vitro. Overlap of the genes identified by RNA-seq and DAP-seq revealed 188 genes that were differentially regulated by OsMYB80 and also carried an OsMYB80-interacting DNA element in the promoter. Ten of these promoter elements were randomly selected for gel shift assay and yeast one-hybrid assay, and all showed OsMYB80 binding. The 10 promoters also showed OsMYB80-dependent induction when co-expressed in rice protoplast. Functional annotation of the 188 genes suggested that OsMYB80 regulates male fertility by directly targeting multiple biological processes. The identification of these genes significantly enriched the gene networks governing anther development and provided much new information for the understanding of pollen development and male fertility.

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Unraveling the Mysteries of Phospholipid Scrambling

Thrombosis and Haemostasis ◽

10.1055/s-0037-1616224 ◽

2001 ◽

Vol 86 (07) ◽

pp. 266-275 ◽

Cited By ~ 135

Author(s):

Therese Wiedmer ◽

Peter Sims

Keyword(s):

Plasma Membrane ◽

Protein Interactions ◽

Cell Activation ◽

Phosphorylation Site ◽

Reticuloendothelial System ◽

Phospholipid Scramblase ◽

New Family ◽

Receptor Sites ◽

Cell Clearance

SummaryPlasma membrane phospholipid asymmetry is maintained by an aminophospholipid translocase that transports phosphatidylserine (PS) and phosphatidylethanolamine (PE) from outer to inner membrane leaflet. Cell activation or injury leads to redistribution of all major lipid classes within the plasma membrane, resulting in surface exposure of PS and PE. Cell surface-exposed PS can serve as receptor sites for coagulation enzyme complexes, and contributes to cell clearance by the reticuloendothelial system. The mechanism(s) by which this PL ”scrambling” occurs is poorly understood. A protein called phospholipid scramblase (PLSCR1) has been cloned that exhibits Ca2+-activated PL scrambling activity in vitro. PLSCR1 belongs to a new family of proteins with no apparent homology to other known proteins. PLSCR1 is palmitoylated and contains a potential protein kinase C phosphorylation site. It further contains multiple PxxP and PPxY motifs, representing potential binding motifs for SH3 and WW domains implicated in mediating protein-protein interactions. Although at least two proteins have been shown to associate with PLSCR1, the functional significance of such interaction remains to be elucidated. Evidence that PLSCR1 may serve functions other than its proposed activity as PL scramblase is also presented.

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Testase 1 (ADAM 24) a plasma membrane-anchored sperm protease implicated in sperm function during epididymal maturation or fertilization

Journal of Cell Science ◽

10.1242/jcs.114.9.1787 ◽

2001 ◽

Vol 114 (9) ◽

pp. 1787-1794 ◽

Cited By ~ 1

Author(s):

G.Z. Zhu ◽

D.G. Myles ◽

P. Primakoff

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Acrosome Reaction ◽

Phosphorylation Site ◽

Equatorial Region ◽

Proteolytic Processing ◽

Mature Sperm ◽

Kinase C

Plasma membrane-anchored proteases have key roles in cell signaling, migration and refashioning the cell surface and its surroundings. We report the first example of a plasma membrane-anchored protease on mature sperm, testase 1 (ADAM 24). Unlike other studied sperm ADAMs (fertilin (α) and (β), cyritestin) whose metalloprotease domains are removed during sperm development, we found testase 1 retains an active metalloprotease domain, suggesting it acts as a protease on mature sperm. Testase 1 is a glycoprotein (molecular mass 88 kDa), localized to the equatorial region of the plasma membrane of cauda epididymal sperm. Typically, proteolytic removal of the pro-domain is an initial activation step for ADAM proteases. The pro-domain of the testase 1 precursor (108 kDa) is proteolytically removed as sperm transit the caput epididymis to produce processed (mature) testase 1 (88 kDa). Testase 1 is unique among all studied ADAMs in that its proteolytic processing occurs on the sperm plasma membrane instead of at an intracellular site (the Golgi). Using GST-fusion proteins and a synthetic testase 1 C-terminal peptide, we found that the cytoplasmic tail of testase 1 could be phosphorylated in vitro by protein kinase C (PKC). Thus testase 1 apparently has a cytoplasmic PKC phosphorylation site(s). Protein kinase C is known to stimulate other ADAMs' protease activity. Because events of the acrosome reaction include PKC activation, we speculate that testase 1 protease function could be important in sperm penetration of the zona pellucida after sperm PKC is activated during the acrosome reaction.

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Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells.

The Journal of Cell Biology ◽

10.1083/jcb.133.2.247 ◽

1996 ◽

Vol 133 (2) ◽

pp. 247-256 ◽

Cited By ~ 165

Author(s):

T Yoshimori ◽

P Keller ◽

M G Roth ◽

K Simons

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Cho Cells ◽

Semliki Forest Virus ◽

Mdck Cells ◽

Wild Type ◽

Vitro Assay ◽

Delivery Routes ◽

The One

The question of how membrane proteins are delivered from the TGN to the cell surface in fibroblasts has received little attention. In this paper we have studied how their post-Golgi delivery routes compare with those in epithelia] cells. We have analyzed the transport of the vesicular stomatitis virus G protein, the Semliki Forest virus spike glycoprotein, both basolateral in MDCK cells, and the influenza virus hemagglutinin, apical in MDCK cells. In addition, we also have studied the transport of a hemagglutinin mutant (Cys543Tyr) which is basolateral in MDCK cells. Aluminum fluoride, a general activator of heterotrimeric G proteins, inhibited the transport of the basolateral cognate proteins, as well as of the hemagglutinin mutant, from the TGN to the cell surface in BHK and CHO cells, while having no effect on the surface delivery of the wild-type hemagglutinin. Only wild-type hemagglutinin became insoluble in the detergent CHAPS during transport through the BHK and CHO Golgi complexes, whereas the basolateral marker proteins remained CHAPS-soluble. We also have developed an in vitro assay using streptolysin O-permeabilized BHK cells, similar to the one we have previously used for analyzing polarized transport in MDCK cells (Pimplikar, S.W., E. Ikonen, and K. Simons. 1994. J. Cell Biol. 125:1025-1035). In this assay anti-NSF and rab-GDI inhibited transport of Semliki Forest virus spike glycoproteins from the TGN to the cell surface while having little effect on transport of the hemagglutinin. Altogether these data suggest that fibroblasts have apical and basolateral cognate routes from the TGN to the plasma membrane.

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Targeting RyR2 with a phosphorylation site-specific nanobody Reverses Dysfunction of Failing Cardiomyocytes in Rat

10.1101/435875 ◽

2018 ◽

Author(s):

Tian Li ◽

Yafeng Shen ◽

Fangxing Lin ◽

Wenyan Fu ◽

Shuowu Liu ◽

...

Keyword(s):

Heart Failure ◽

Gene Transfer ◽

Gene Delivery ◽

Phosphorylation Site ◽

Ischemic Heart ◽

Ischemic Heart Failure ◽

Adeno Associated Virus ◽

Vitro Assay ◽

Site Specific

AbstractChronic PKA phosphorylation of RyR2 has been shown to increased diastolic SR Ca2+ leak and lead to cardiac dysfunction. Since the change of phosphorylation level of RyR2 is a biomarker of failing heart, we attempted to verify the hypothesis that intracellular gene delivery of a RyR2 targeting phosphorylation site-specific nanobody could preserve contractility of failing myocardium. In present study, we acquired the RyR2-specific nanobodies from a phage display library which are variable domains of camellidae heavy chain-only antibodies (VHH). One of the monoclonal nanobodies, AR185, inhibiting RyR2 phosphorylation in an in vitro assay was then chosen for further investigation. We investigated the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of AR185 against post-ischemic heart failure. Adeno-associated virus gene delivery elevated the intracellular expression AR185 protein in the ischemic heart failure model of rats, and this treatment normalized the systolic and diastolic dysfunction of the failing myocardium in vivo and in vitro by reversing myocardial Ca2+ handling. Furthermore, AR185 gene transfer to failing cardiomyocytes reduced the frequency of sarcoplasmic reticulum (SR) calcium leak, thereby restoring the attenuated intracellular calcium transients and SR calcium load. Moreover, AR185 gene transfer inhibited PKA phosphorylation of RyR2 in failing cardiomyocytes. Our results provided strong pre-clinical experimental evidence of the cardiac expression of RyR2 nanobody with AAV9 vectors as a promising therapeutic strategy for ischemic heart failure.

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SNAP-23 Functions in Docking/Fusion of Granules at Low Ca2+

Molecular Biology of the Cell ◽

10.1091/mbc.e03-09-0684 ◽

2004 ◽

Vol 15 (4) ◽

pp. 1918-1930 ◽

Cited By ~ 41

Author(s):

Evelina Chieregatti ◽

Michael C. Chicka ◽

Edwin R. Chapman ◽

Giulia Baldini

Keyword(s):

Plasma Membrane ◽

Endocrine Cells ◽

Secretory Granules ◽

Hormone Secretion ◽

Plasma Membrane Protein ◽

Vitro Assay ◽

Functional Differences ◽

Different Levels ◽

Granule Release

Ca2+-triggered exocytosis of secretory granules mediates the release of hormones from endocrine cells and neurons. The plasma membrane protein synaptosome-associated protein of 25 kDa (SNAP-25) is thought to be a key component of the membrane fusion apparatus that mediates exocytosis in neurons. Recently, homologues of SNAP-25 have been identified, including SNAP-23, which is expressed in many tissues, albeit at different levels. At present, little is known concerning functional differences among members of this family of proteins. Using an in vitro assay, we show here that SNAP-25 and SNAP-23 mediate the docking of secretory granules with the plasma membrane at high (1 μM) and low (100 nM) Ca2+ levels, respectively, by interacting with different members of the synaptotagmin family. In intact endocrine cells, expression of exogenous SNAP-23 leads to high levels of hormone secretion under basal conditions. Thus, the relative expression levels of SNAP-25 and SNAP-23 might control the mode (regulated vs. basal) of granule release by forming docking complexes at different Ca2+ thresholds.

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Absence of SCAPER causes male infertility in humans and Drosophila by modulating microtubule dynamics during meiosis

Journal of Medical Genetics ◽

10.1136/jmedgenet-2020-106946 ◽

2020 ◽

pp. jmedgenet-2020-106946 ◽

Cited By ~ 1

Author(s):

Ohad Wormser ◽

Ygal Levy ◽

Anna Bakhrat ◽

Silvia Bonaccorsi ◽

Lucia Graziadio ◽

...

Keyword(s):

Total Internal Reflection ◽

Testicular Tissue ◽

Male Meiosis ◽

Testicular Sperm Extraction ◽

Male Sterile ◽

Vitro Assay ◽

Clinical Phenotyping ◽

Null Mutants ◽

Variable Penetrance

BackgroundMutation in S-phase cyclin A-associated protein rin the endoplasmic reticulum (SCAPER) have been found across ethnicities and have been shown to cause variable penetrance of an array of pathological traits, including intellectual disability, retinitis pigmentosa and ciliopathies.MethodsHuman clinical phenotyping, surgical testicular sperm extraction and testicular tissue staining. Generation and analysis of short spindle 3 (ssp3) (SCAPER orthologue) Drosophila CAS9-knockout lines. In vitro microtubule (MT) binding assayed by total internal reflection fluorescence microscopy.ResultsWe show that patients homozygous for a SCAPER mutation lack SCAPER expression in spermatogonia (SPG) and are azoospermic due to early defects in spermatogenesis, leading to the complete absence of meiotic cells. Interestingly, Drosophila null mutants for the ubiquitously expressed ssp3 gene are viable and female fertile but male sterile. We further show that male sterility in ssp3 null mutants is due to failure in both chromosome segregation and cytokinesis. In cells undergoing male meiosis, the MTs emanating from the centrosomes do not appear to interact properly with the chromosomes, which remain dispersed within dividing spermatocytes (SPCs). In addition, mutant SPCs are unable to assemble a normal central spindle and undergo cytokinesis. Consistent with these results, an in vitro assay demonstrated that both SCAPER and Ssp3 directly bind MTs.ConclusionsOur results show that SCAPER null mutations block the entry into meiosis of SPG, causing azoospermia. Null mutations in ssp3 specifically disrupt MT dynamics during male meiosis, leading to sterility. Moreover, both SCAPER and Ssp3 bind MTs in vitro. These results raise the intriguing possibility of a common feature between human and Drosophila meiosis.

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Casein Kinase 2 Binds to the C Terminus of Na+/H+ exchanger 3 (NHE3) and Stimulates NHE3 Basal Activity by Phosphorylating a Separate Site in NHE3

Molecular Biology of the Cell ◽

10.1091/mbc.e08-01-0019 ◽

2008 ◽

Vol 19 (9) ◽

pp. 3859-3870 ◽

Cited By ~ 38

Author(s):

Rafiquel Sarker ◽

Mads Grønborg ◽

Boyoung Cha ◽

Sachin Mohan ◽

Yueping Chen ◽

...

Keyword(s):

Plasma Membrane ◽

Phosphorylation Site ◽

Casein Kinase ◽

Casein Kinase 2 ◽

Kinase Assay ◽

Phosphatase Inhibitors ◽

C Terminus ◽

Separate Site ◽

A Site

Na+/H+ exchanger 3 (NHE3) is the epithelial-brush border isoform responsible for most intestinal and renal Na+ absorption. Its activity is both up- and down-regulated under normal physiological conditions, and it is inhibited in most diarrheal diseases. NHE3 is phosphorylated under basal conditions and Ser/Thr phosphatase inhibitors stimulate basal exchange activity; however, the kinases involved are unknown. To identify kinases that regulate NHE3 under basal conditions, NHE3 was immunoprecipitated; LC-MS/MS of trypsinized NHE3 identified a novel phosphorylation site at S719 of the C terminus, which was predicted to be a casein kinase 2 (CK2) phosphorylation site. This was confirmed by an in vitro kinase assay. The NHE3-S719A mutant but not NHE3-S719D had reduced NHE3 activity due to less plasma membrane NHE3. This was due to reduced exocytosis plus decreased plasma membrane delivery of newly synthesized NHE3. Also, NHE3 activity was inhibited by the CK2 inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole DMAT when wild-type NHE3 was expressed in fibroblasts and Caco-2 cells, but the NHE3-S719 mutant was fully resistant to DMAT. CK2 bound to the NHE3 C-terminal domain, between amino acids 590 and 667, a site different from the site it phosphorylates. CK2 binds to the NHE3 C terminus and stimulates basal NHE3 activity by phosphorylating a separate single site on the NHE3 C terminus (S719), which affects NHE3 trafficking.

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Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells

AJP Renal Physiology ◽

10.1152/ajprenal.1997.272.6.f816 ◽

1997 ◽

Vol 272 (6) ◽

pp. F816-F822 ◽

Cited By ~ 92

Author(s):

T. Katsura ◽

C. E. Gustafson ◽

D. A. Ausiello ◽

D. Brown

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Protein Kinase A ◽

Phosphorylation Site ◽

Transfected Cells ◽

Aquaporin 2 ◽

Intracellular Vesicles ◽

Kinase A

Vasopressin-dependent translocation of aquaporin-2 (AQP2) between intracellular vesicles and the plasma membrane has been demonstrated in vivo and in vitro. Furthermore, the vasopressin-induced increase in apical membrane water permeability of renal principal cells is dependent on a rise in intracellular adenosine 3',5'-cyclic monophosphate and activation of protein kinase A (PKA). To determine whether trafficking of AQP2 is dependent on PKA phosphorylation, we first examined the effect of the PKA-inhibitor N-(2[[3-(4-bromophenyl)-2-propenyl]-amino]-ethyl)-5-isoquinolinesulfonam ide (H-89) on AQP2 translocation in transfected LLC-PK1 cells. Vasopressin-induced membrane insertion of AQP2 was completely inhibited by pretreatment of the cells for 60 min with H-89. This reagent also caused a dense accumulation of AQP2 in the Golgi region. Next, LLC-PK1 cells were stably transfected with AQP2 cDNA in which the PKA phosphorylation site, Ser256, was replaced with alanine (S256A). S256A-AQP2 was not phosphorylated in vitro by PKA, and S256A-AQP2 was mainly localized to intracellular vesicles in the basal condition, similar to wild-type AQP2. However, after stimulation with vasopressin or forskolin, the cellular distribution of S256A-AQP2 remained unchanged. In addition, the usual vasopressin-induced increase in endocytosis seen in AQP2-transfected cells was not observed in S256A-AQP2-transfected cells. These results demonstrate that the Ser256 PKA phosphorylation site is possibly involved in the vasopressin-induced trafficking of AQP2 from intracellular vesicles to the plasma membrane and in the subsequent stimulation of endocytosis.

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An L-type lectin receptor-like kinase promotes starch accumulation during rice pollen maturation

Development ◽

10.1242/dev.196378 ◽

2021 ◽

pp. dev.196378

Author(s):

Zhiyuan He ◽

Ting Zou ◽

Qiao Xiao ◽

Guoqiang Yuan ◽

Miaomiao Liu ◽

...

Keyword(s):

Starch Synthesis ◽

Pollen Grains ◽

Starch Accumulation ◽

Male Sterile ◽

Pollen Maturation ◽

Lectin Receptor ◽

Rice Mutant ◽

Receptor Like Kinase ◽

Mutant Pollen

Starch accumulation is key for the maturity of rice pollen grains; however, the regulatory mechanism underlying this process remains unknown. Here, we isolated a male-sterile rice mutant, abnormal pollen 1 (ap1), which produces nonviable pollen grains with defective starch accumulation. Functional analysis revealed that AP1 encodes an active L-type lectin receptor-like kinase (L-LecRLK). AP1 is localized to the plasma membrane and its transcript is highly accumulated in pollen during the starch synthesis phase. RNA-seq and phosphoproteomic analysis revealed that the expression/phosphorylation levels of numerous genes/proteins involved in starch and sucrose metabolism pathway were significantly altered in the mutant pollen, including a known rice UDP-glucose pyrophosphorylase (OsUGP2). We further found that AP1 physically interacts with OsUGP2 to elevate its enzymatic activity likely through targeted phosphorylation. These findings revealed a novel role of L-LecRLK in controlling pollen maturity via modulating sucrose and starch metabolism.

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