ABCMdb

ABCC2 p.Ser914Ala

Predicted by SNAP2:	A: N (66%), C: D (53%), D: N (57%), E: N (66%), F: D (53%), G: N (72%), H: N (53%), I: N (57%), K: N (66%), L: N (61%), M: N (53%), N: N (78%), P: N (61%), Q: N (78%), R: N (72%), T: N (78%), V: N (66%), W: D (63%), Y: D (59%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: D, I: N, K: N, L: D, M: N, N: N, P: D, Q: N, R: N, T: N, V: N, W: D, Y: D,

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[hide] Suppression of Ycf1p function by Cka1p-dependent p... FEMS Yeast Res. 2010 Nov;10(7):839-57. doi: 10.1111/j.1567-1364.2010.00677.x. Epub 2010 Aug 31. Pickin KA, Ezenwajiaku N, Overcash H, Sethi M, Knecht MR, Paumi CM
Suppression of Ycf1p function by Cka1p-dependent phosphorylation is attenuated in response to salt stress.
FEMS Yeast Res. 2010 Nov;10(7):839-57. doi: 10.1111/j.1567-1364.2010.00677.x. Epub 2010 Aug 31., [PMID:20812950]

Abstract [show]
The yeast vacuolar membrane protein Ycf1p and its mammalian counterpart, MRP1, belong to the ABCC subfamily of ATP-binding cassette transporters. Genetic evidence suggests that the yeast casein kinase 2alpha, Cka1p, negatively regulates Ycf1p function via phosphorylation of Ser251 within the N-terminus. In this study, we provide strong evidence that Cka1p regulates Ycf1p function via phosphorylation of Ser251. We show that the CK2 holoenzyme interacts with Ycf1p. However, genetic analysis suggests that only Cka1p is required for Ser251 phosphorylation, as the deletion of CKA1 significantly reduces Ser251 phosphorylation in vivo. Furthermore, purified recombinant Cka1p phosphorylates a Ycf1p-derived peptide containing Ser251. We also demonstrate that Ycf1p function is induced in response to high salt stress. Induction of the Ycf1p function strongly correlates with reduced phosphorylation of Ser251. Importantly, Cka1p activity in vivo is similarly reduced in response to salt stress, consistent with our finding that Cka1p directly phosphorylates Ser251 of Ycf1p. We provide genetic and biochemical evidence that strongly suggests that the induction of Ycf1p function is the result of decreased phosphorylation of Ser251. In conclusion, our work demonstrates a novel biochemical role for Cka1p regulation of Ycf1p function in the cellular response of yeast to salt stress.

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73 PCR-mediated mutagenesis, followed by homologous recombination resulted in the generation of pCP83, pCP84, pCP85, pCP94, pCP95, and pCP96 (YCF1 mutant plasmids Ser869Ala, Ser903Ala, Ser914Ala, Ser870Ala, Ser872Ala, and Ser873Ala, respectively). Login to comment
84 Yeast strains used in this study StrainÃ Relevant genotype Reference CP59w met3Dleu2Dura3Dhis3D Open biosystems CP60 met3Dleu2Dura3Dhis3Dycf1<KanMX Open biosystems CP105 ura3Dade2DLEU2 TRP1 YCF1 This study CP106 ura3DADE2 LEU2 TRP1 YCF1 This study CP135 met15Dleu2Dura3Dhis3Dycf1D<KanMx [2m YCF1-GFP URA3] This study CP136 met15Dleu2Dura3Dhis3Dycf1D<KanMx [2m YCF1-Ser251Ala -GFP URA3] This study CP147 met15Dleu2Dura3Dhis3Dcka1D<KanMx Open biosystems CP151 met15Dleu2Dura3Dhis3Dcka1D<KanMX [2m YCF1-GFP URA3] This study CP156 met15Dleu2Dura3Dhis3DYCF1:TAP-HIS3 Open biosystems CP157 met15Dleu2Dura3Dhis3Dycf11D<KanMX cka1D<URA3 Paumi et al. (2009) CP159 met15Dleu2Dura3Dhis3Dcka2D<KanMX Open biosystems CP164 met15Dleu2Dura3Dhis3DYCF1:TAP-HIS3 KanMx-PGAL-GST-CKA1 This study CP165 met15Dleu2Dura3Dhis3Dycf1D<KanMX [2m YCF1-S869A-GFP URA3] This study CP166 met15Dleu2Dura3Dhis3Dycf1D<KanMX [2m YCF1-S870A -GFP URA3] This study CP167 met15Dleu2Dura3Dhis3Dycf1D<KanMX [2m YCF1-S872A -GFP URA3] This study CP168 met15Dleu2Dura3Dhis3Dycf1D<KanMX [2m YCF1-S873A-GFP URA3] This study CP169 met15Dleu2Dura3Dhis3Dycf1D<KanMX [2m YCF1-S903A-GFP URA3] This study CP170 met15Dleu2Dura3Dhis3Dycf1D<KanMX [2m YCF1-S914A-GFP URA3] This study CP174 PYES2/CT<cka1<v5<6his Kubinski et al. (2007) CP176 met15Dleu2Dura3Dhis3Dcka2D<KanMX [2m CKA2:MYC:6xHis LEU2] This study CP177 met15Dleu2Dura3Dhis3DpepD<KanMX [2m YCF1-GFP URA3] This study CP178 met15Dleu2Dura3Dhis3Dcka2D<KanMX [2m YCF1-GFP URA3] This study CP179 met15Dleu2Dura3Dhis3DKanMx-PGAL-GST-CKA1 This study CP180 met15Dleu2Dura3Dhis3DpepD<KanMX [2m YCF1-Ser251Ala-GFP URA3] This study CP187 met15Dleu2Dura3Dhis3Dcka1D<LEU2 pep4D<KanMX [2m YCF1-TAP URA3] This study CP188 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-TAP URA3] This study CP189 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-Ser869Ala-GFP URA3] This study CP190 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-Ser870Ala-GFP URA3] This study CP191 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-Ser872Ala-GFP URA3] This study CP192 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-Ser873Ala-GFP URA3] This study CP193 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-Ser903Ala-GFP URA3] This study CP194 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-Ser914Ala-GFP URA3] This study CP195 met15Dleu2Dura3Dhis3DCKA1-V5-His<KanMx Open biosystems CP200 met15Dleu2Dura3Dhis3Dpep4D<KanMX [2m YCF1-Ser908Ala,Thr911Ala-GFP URA3] This study CP224 met15Dleu2Dura3Dhis3Dckb1D<KanMX Open biosystems CP225 met15Dleu2Dura3Dhis3Dckb2D<KanMX Open biosystems CP226 met15Dleu2Dura3Dhis3DYCF1:TAP-HIS3 [2m PGAL-GST-CKA2 URA3] This Study CP227 met15Dleu2Dura3Dhis3DYCF1:TAP-HIS3 [2m PGAL-GST-CKB1 URA3] This study CP228 met15Dleu2Dura3Dhis3DYCF1:TAP-HIS3 [2m PGAL-GST-CKB2 URA3] This study CP229 met15Dleu2Dura3Dhis3Dckb1D<KanMX [2m YCF1-GFP URA3] This study CP230 met15Dleu2Dura3Dhis3Dckb2D<KanMX [2m YCF1-GFP URA3] This study CP239 met3Dleu2Dura3Dhis3Dycf1<KanMX [2m YCF1 -GFP URA3] This study CP240 met3Dleu2Dura3Dhis3Dycf1<KanMX [2m YCF1 -Ser251Ala-GFP URA3] This study CP241 met3Dleu2Dura3Dhis3Dycf1<KanMX [2m YCF1 -Ser251Glu-GFP URA3] This study ÃAll CP strains listed here are isogenic to CP59. Login to comment
103 Plasmid used in this study Plasmid Relevant genotype Reference pCP25 (pFA6) KanMx6-PGAL1-GST Longtine et al. (1998) pCP50 (pSM2243) [2m YCF1-Ser251Aala-GFP URA3] Paumi et al. (2008) pCP58 (pSM1753) [2m YCF1-GFP URA3] Mason & Michaelis (2002) pCP74 [2m CKA2:MYC:6xHIS LEU2] Schaerer-Brodbeck & Riezman (2003) pCP83 [2m YCF1-Ser869Ala-GFP URA3] This study pCP84 [2m YCF1-Ser903Ala-GFP URA3] This study pCP85 [2m YCF1-Ser914Ala-GFP URA3] This study pCP94 [2m YCF1-Ser870Ala-GFP URA3] This study pCP95 [2m YCF1-Ser872Ala-GFP URA3] This study pCP96 [2m YCF1-Ser873Ala-GFP URA3] This study pCP109 [2m PGAL-GST-CKA2 URA3] Open biosystems pCP110 [2m PGAL-GST-CKB1 URA3] Open biosystems pCP111 [2m PGAL-GST-CKB2 URA3] Open biosystems transported from outside to inside the vesiculated vacuoles. Login to comment
162 It is worth noting that Ycf1p-Ser873Ala and Ycf1p-Ser914Ala have a very minimal, yet notable, increased resistance to the low concentration of cadmium (50 mM) (Fig. 2a, 2nd column, compare rows 1 and 7 and 1 and 9). Login to comment
174 Cka1p is required for the phosphorylation of Ycf1p-Ser251 in vivo Although the study by Paumi et al. (2008) provides substantial support for the direct phosphorylation of Ycf1p by Cka1p, the direct phosphorylation of Ser251 by Cka1p has WT ycf1 YCF1-S869A YCF1-S903A YCF1-S914A (a) WT BF FITC (b) α-GFP β-Actin 150 kDa 250 kDa 36 kDa 50 kDa Relative expression (c) S903A S914AS872A S873AS869A S870A 0 50 100 150 200 1molofE2B17G 10min-1mg-1 (d) 0 μM CdSO 200 μM CdSO50 μM CdSO YCF1-S251A YCF1-S870A YCF1-S872A YCF1-S873A S251A 1 0 0.9 1 0.9 1.2 1.1 0.91 0 0.85 Fig. 2. Login to comment
338 It is important to note, however, that Ycf1p-Ser873Ala and Ycf1p-Ser914Ala show a subtle increase in resistance over that of WT at a low concentration of cadmium. Login to comment