Long T et al. (2012), SmSak, the second Polo-like kinase of the helmi...

XB-ART-45593

PLoS One 2012 Jan 01;76:e40045. doi: 10.1371/journal.pone.0040045.

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SmSak, the second Polo-like kinase of the helminth parasite Schistosoma mansoni: conserved and unexpected roles in meiosis.

Long T , Vanderstraete M , Cailliau K , Morel M , Lescuyer A , Gouignard N , Grevelding CG , Browaeys E , Dissous C .

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Polo-like kinases (Plks) are a family of conserved regulators of a variety of events throughout the cell cycle, expanded from one Plk in yeast to five Plks in mammals (Plk1-5). Plk1 is the best characterized member of the Plk family, homolog to the founding member Polo of Drosophila, and plays a major role in cell cycle progression by triggering G2/M transition. Plk4/Sak (for Snk (Serum-inducible kinase) akin kinase) is a unique member of the family, structurally distinct from other Plk members, with essential functions in centriole duplication. The genome of the trematode parasite Schistosoma mansoni contains only two Plk genes encoding SmPlk1 and SmSak. SmPlk1 has been shown already to be required for gametogenesis and parasite reproduction. In this work, in situ hybridization indicated that the structurally conserved Plk4 protein, SmSak, was largely expressed in schistosome female ovary and vitellarium. Expression of SmSak in Xenopus oocytes confirmed its Plk4 conserved function in centriole amplification. Moreover, analysis of the function of SmSak in meiosis progression of G2-blocked Xenopus oocytes indicated that, in contrast to SmPlk1, SmSak cannot induce G2/M transition in the absence of endogenous Plk1 (Plx1). Unexpectedly, meiosis progression was spontaneously observed in Plx1-depleted oocytes co-expressing SmSak and SmPlk1. Molecular interaction between SmSak and SmPlk1 was confirmed by co-immunoprecipitation of both proteins. These data indicate that Plk1 and Plk4 proteins have the potential to interact and cross-activate in cells, thus attributing for the first time a potential role of Plk4 proteins in meiosis/mitosis entry. This unexpected role of SmSak in meiosis could be relevant to further consider the function of this novel Plk in schistosome reproduction.

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Species referenced: Xenopus
Genes referenced: cdc25c cer1 crygdl.43 myc pbrm1 plk1 plk4 rasgrf1 synpr

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	Figure 1. Structure of SmPlk1 and SmSak proteins.Plk1 and Sak proteins possess an amino-terminal serine/threonine kinase domain and a carboxy-terminal polo-box domain (PBD) that dictates the substrate specificity of the enzymes and regulates their function. Phosphorylation of a specific threonine (T182 or T175 respectively in SmPlk1 or SmSak) in the T-loop activation domain determines the activity of the kinases. Plk1 PBD is made up of two polo-boxes, PB1 and PB2, that form intramolecular heterodimers able to bind specific phospho-motifs. Binding to phosphosubstrates induces conformational changes and the access of the catalytic domain to its substrates [5], [6]. Both PB1(AA 386â453) and PB2 (AA 488â557) are contained in the sequence of SmPlk1. Sak PBD contains only one C-terminal PB, and a larger cryptic polo-box (cry-pb) weakly homologous to PB and unable to heterodimerize intramolecularly. However, PB from two Sak molecules can homodimerize and be involved in the regulation of the kinase activity. SmSak possesses a cry-pb (AA 533â802) and one PB (AA 807â896) containing a PEST destruction motif (AA 853â866) implicated very likely in the regulation of its turnover.
	Figure 2. Quantification of SmSak transcripts in the parasite stages.A. SmSak transcripts were quantified in the different stages of the parasite S. mansoni (M, male; F, female; Mir, miracidia; Spo, sporocyst; Cer, cercaria; Sch, schistosomulum). Tubulin transcript level was used as internal standard. For graphical representation of qPCR data, ÎCt values calculated for SmSak in each parasitic stage were compared to the ÎCt value obtained for males using the deltadeltaCt (ÎÎCt) method [65]. Values were normalized as fold-difference relative to males. Mean of three determinations +/â SD. B. SmSak/SmPlk1 ratios in each parasite stage. SmSak and SmPlk1 transcripts were both quantified in two independent cDNA fractions prepared from each stage. In this case, SmPlk1 was used as a reference. Mean values of ratios +/â SD are given.
	Figure 3. In situ localization of transcripts in adult worms.In situ hybridization experiments using DIG-labelled antisense- (AâCâE) and sense-RNA probes (BâDâF) of SmSak. SmSak transcripts were localized mainly in female reproductive organs: vitellarium and ovary in female worms (AâCâE) and to a lesser extent, in the testes of male worms (A). Scale barsâ=â100 Âµm (AâBâEâF), 50 Âµm (C, D). Abbreviations: te, testes; o, ovary; io, immature ovary; mo, mature ovary; v, vitellarium.
	Figure 4. Analysis of SmSak activity in Xenopus oocytes.A. Constitutively active SmSak induces germinal vesicle breakdown (GVBD) and meiosis progression in Xenopus oocytes. Different samples of oocytes (nâ=â15) were microinjected with cRNA encoding SmSak, SmSakDK, SmSakT175D, SmSakT175DDK or SmSakT175A. Percentages of oocytes exhibiting GVBD 18 h after microinjection of cRNA are indicated. Non-injected oocytes were used as control. Western blot analysis of oocyte extracts was performed as described in materials and methods to detect the gel-shift of endogenous phosphorylated Plx1 and Cdc25C proteins (indicated by arrows). The expression of the different forms of V5-tagged recombinant SmSak was confirmed by the use of anti-V5 antibodies. B. SmSak mutants do not alter the GVBD induced by progesterone. The experiment was performed as in A, except that PG was added to the different oocyte groups two hours after microinjection of cRNA preparations. Gel-shifts of Plx1 and Cdc25C are observed in all oocyte groups associated with high levels of GVBD. C. Endogenous Plx1 is required for the induction of GVBD by SmSakT175D. Oocytes were injected or not with 5 ng anti-Plx1 antibodies one hour before the injection of 10 or 60 ng SmSak175D cRNA. Meiosis resumption was monitored as in A and B. SmSakT175D induces GVBD (as already shown in A) following the injection of 60 ng cRNA but has no more effect in Plx1-depleted oocytes. In control oocytes, it was confirmed that anti-Plk1 antibodies blocked completely the GVBD induced by PG. D. Comparative analysis of the capacity of SmSak and SmPlk1 to induce GVBD in different conditions. Normal versus Plx1-depleted oocytes were incubated with or without (w/o) PG following the expression of SmPlk1, SmPlkT182D, SmSak or SmSakT175D. PG stimulates GVBD in the oocytes expressing each of the parasite kinases, provided that Plx1 is functional in oocytes. In Plx1-depleted oocytes, only SmPlk1 proteins are efficient. In the absence of PG, only constitutively active kinases are efficient, provided that Plx1 is functional. In Plx1-depleted oocytes, only SmPlkT182D can induce GVBD. These results were correlated with our previous data [23] and those presented in A, B and C. All percentages of GVBD represent the mean of three independent experiments. In A, B and C, blots are representative for the three experiments.
	Figure 5. SmSak induces de novo centriole formation in in vitro matured Xenopus oocytes.Samples of cRNA encoding SmSak, SmSakT175A, SmPlk1 or Kin1-PBD4 and Kin4-PBD1 hybrid proteins, were microinjected in oocytes and incubated for 2 h for protein expression, before addition of PG. At 15 h following the addition of PG, GVBD monitored by the formation of a white spot centered at the germinal pole of Metaphase II arrested oocytes, was observed in all oocyte groups. Matured oocytes were then activated with calcium ionophore A23187, and surface morphology was observed after 30 min or 2 h. Only SmSak-expressing oocytes formed pigmented speckles that increased in number from 30 min to 2 h. In all other oocytes, the animal pole gradually darkened after the addition of A23187 and the formation of a black spot (fertilization coat) could be seen.
	Figure 6. Studies of the importance of SmPlk1 and SmSak domains for oocyte maturation by using SmPlk1/SmSak hybrid constructs.A. Schematic representation of Kin1-PBD4 and Kin4-PBD1 hybrids formed by the fusion of the N-terminal part of SmPlk1 (AA 1â342) with the C-terminal SmSak (AA 323â618) and that of N-terminal SmSak (AA 1â322) with C-terminal SmPlk1 (AA 343â903), respectively (refer to Figure 1). B. Different sets of Plx1-depleted oocytes were microinjected with cRNA encoding SmPlk1, SmSak, Kin1-PBD4 or Kin4-PBD1, or their unphosphorylable variants, and stimulated by the addition of PG. GVBD monitored 18 h after microinjection of cRNA, only occurred in oocytes expressing SmPlk1 and Kin4-PBD1 that contain phosphorylable T182 and T175, respectively. Western blot analysis of oocyte extracts with anti-V5 antibodies confirmed the expression of proteins in oocytes. Gel shift of Cdc25 was detected only in matured oocytes.
	Figure 7. Interaction between SmPlk1 and SmSak and kinase co-activation.A. Co-immunoprecipitation of SmPlk1 and SmSak expressed in Xenopus oocytes. Oocytes were injected with V5 tagged-SmPlk1, Myc tagged-SmSak or with both kinases simultaneously. Non-injected oocytes were used as controls. Following 18 h, GVBD was monitored. Oocyte lysates were prepared and parasite kinases were immunoprecipitated with anti-V5 or anti-Myc antibodies, then analyzed by western blotting. Results show that immunoprecipitates contained both SmPlk1 and SmSak when the proteins were co-expressed. B. Co-activation of SmPlk1 and SmSak induces GVBD. In normal or Plx1-depleted oocytes, SmSak was expressed with SmPlk1 or with SmPlk1T182V and reciproqually SmPlk1 was expressed with SmSak or with SmSak T175A. SmPlk1T182V and SmSakT175A were also expressed together. Only the co-expression of SmSak and SmPlk1 induced GVBD in normal as well as in Plx1-depleted oocytes. All the other combinations were inefficient, except the co-expression of SmSak with SmPlk1T182V, that allowed GVBD but only in normal oocytes (containing a functional Plx1).

References [+] :

Andrysik, The novel mouse Polo-like kinase 5 responds to DNA damage and localizes in the nucleolus. 2010, Pubmed