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Casein kinase 1 controls the activation threshold of an α-arrestin by multisite phosphorylation of the interdomain hinge.

Herrador A, Livas D, Soletto L, Becuwe M, Léon S, Vincent O - Mol. Biol. Cell (2015)

Bottom Line: We show that Rim8 is phosphorylated in a pH-independent but Rim21-dependent manner by the plasma membrane-associated casein kinase 1 (CK1).Our results indicate that Rim8 phosphorylation prevents its accumulation at the plasma membrane at acidic pH and thereby inhibits RIM signaling.These findings support a model in which CK1-mediated phosphorylation of Rim8 contributes to setting a signaling threshold required to inhibit the RIM pathway at acidic pH.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, 28029 Madrid, Spain.

No MeSH data available.


Related in: MedlinePlus

Effect of Rim21 C-tail overexpression on RIM signaling and Rim8 localization and phosphorylation. (A) Phenotypic analysis of rim mutant strains overexpressing Rim21 or Dfg16 C-tails (CT). Y01150 (rim21∆), Y11806 (dfg16∆), Y06196 (rim9∆), and Y04414 (rim8∆) were transformed with pADH1-Flag-Rim21CT, pADH1-Flag-Dfg16CT, or empty vector (none). Growth tests in the presence of LiCl were as described for Figure 1D. Immunoblot analysis of transformants expressing Rim21CT and Dfg16CT confirmed the expression of both proteins (unpublished data). (B) Effect of Rim21 C-tail overexpression on Rim101 processing. OVY24 (wild type [WT] RIM101-HA) and OVY43 (rim21∆ RIM101-HA) were transformed with pADH1-Flag-Rim21CT (+) or empty vector (–). After a shift to pH 7.5, protein extracts were prepared and immunoblotted with anti-HA antibody. (C) Effect of ambient pH on Rim8 localization. OVY172 (RIM8-3GFP) was grown to mid log phase in YPD medium and then injected into a microfluidics device in SD medium containing 0.1 M HEPES, pH 4. Cells were then perfused with SD medium containing 0.1 M HEPES, pH 7, and examined by fluorescence and DIC microscopy before and after the change of medium at the indicated time. (D) Rim8 localization in cells overexpressing Rim21 C-tail. OVY172 (RIM8-3GFP) and OVY304 (rim21∆ RIM8-3GFP) were transformed with pADH1-Flag-Rim21CT or empty vector as indicated on the left. Cells were grown to mid log phase on SD medium (final pH 3.5) and examined by fluorescence and DIC microscopy. (E) Colocalization of Rim8 and overexpressed Rim21 C-tail. OVY172 (RIM8-3GFP) and OVY31(rim8∆) were transformed with pADH1-RFP-Rim21CT, grown to mid log phase on SD medium (final pH 3.5), and examined by fluorescence and DIC microscopy. (F) Effect of Rim21 C-tail overexpression on Rim8 phosphorylation. Y04414 was cotransformed with pHA-Rim8 (top) or a Rim8 truncated derivative (residues 1–460; bottom) and pADH1-Flag-Rim21CT (+) or empty vector (–). Transformants were grown to mid log phase (final pH 3.5), and protein extracts were prepared and immunoblotted with anti-HA antibody.
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Figure 3: Effect of Rim21 C-tail overexpression on RIM signaling and Rim8 localization and phosphorylation. (A) Phenotypic analysis of rim mutant strains overexpressing Rim21 or Dfg16 C-tails (CT). Y01150 (rim21∆), Y11806 (dfg16∆), Y06196 (rim9∆), and Y04414 (rim8∆) were transformed with pADH1-Flag-Rim21CT, pADH1-Flag-Dfg16CT, or empty vector (none). Growth tests in the presence of LiCl were as described for Figure 1D. Immunoblot analysis of transformants expressing Rim21CT and Dfg16CT confirmed the expression of both proteins (unpublished data). (B) Effect of Rim21 C-tail overexpression on Rim101 processing. OVY24 (wild type [WT] RIM101-HA) and OVY43 (rim21∆ RIM101-HA) were transformed with pADH1-Flag-Rim21CT (+) or empty vector (–). After a shift to pH 7.5, protein extracts were prepared and immunoblotted with anti-HA antibody. (C) Effect of ambient pH on Rim8 localization. OVY172 (RIM8-3GFP) was grown to mid log phase in YPD medium and then injected into a microfluidics device in SD medium containing 0.1 M HEPES, pH 4. Cells were then perfused with SD medium containing 0.1 M HEPES, pH 7, and examined by fluorescence and DIC microscopy before and after the change of medium at the indicated time. (D) Rim8 localization in cells overexpressing Rim21 C-tail. OVY172 (RIM8-3GFP) and OVY304 (rim21∆ RIM8-3GFP) were transformed with pADH1-Flag-Rim21CT or empty vector as indicated on the left. Cells were grown to mid log phase on SD medium (final pH 3.5) and examined by fluorescence and DIC microscopy. (E) Colocalization of Rim8 and overexpressed Rim21 C-tail. OVY172 (RIM8-3GFP) and OVY31(rim8∆) were transformed with pADH1-RFP-Rim21CT, grown to mid log phase on SD medium (final pH 3.5), and examined by fluorescence and DIC microscopy. (F) Effect of Rim21 C-tail overexpression on Rim8 phosphorylation. Y04414 was cotransformed with pHA-Rim8 (top) or a Rim8 truncated derivative (residues 1–460; bottom) and pADH1-Flag-Rim21CT (+) or empty vector (–). Transformants were grown to mid log phase (final pH 3.5), and protein extracts were prepared and immunoblotted with anti-HA antibody.

Mentions: Because Rim8 binding to Rim21 plays an essential role in RIM signaling (Figure 1D), we tested whether overexpression of the Rim21 C-tail fragment can bypass the requirement for the pH-sensing machinery in the activation of the RIM pathway. Lithium growth tests indicate that overexpression of Rim21 C-tail, but not Dfg16 C-tail, restores RIM signaling in rim21Δ, dfg16Δ, and rim9Δ mutants but not in a rim8Δ mutant (Figure 3A). Accordingly, Rim21 C-tail overexpression restores Rim101 processing in a rim21Δ mutant (Figure 3B). Thus overexpression of Rim21 C-tail can activate the RIM pathway in the absence of any component of the pH-sensing machinery but still in a Rim8-dependent manner.


Casein kinase 1 controls the activation threshold of an α-arrestin by multisite phosphorylation of the interdomain hinge.

Herrador A, Livas D, Soletto L, Becuwe M, Léon S, Vincent O - Mol. Biol. Cell (2015)

Effect of Rim21 C-tail overexpression on RIM signaling and Rim8 localization and phosphorylation. (A) Phenotypic analysis of rim mutant strains overexpressing Rim21 or Dfg16 C-tails (CT). Y01150 (rim21∆), Y11806 (dfg16∆), Y06196 (rim9∆), and Y04414 (rim8∆) were transformed with pADH1-Flag-Rim21CT, pADH1-Flag-Dfg16CT, or empty vector (none). Growth tests in the presence of LiCl were as described for Figure 1D. Immunoblot analysis of transformants expressing Rim21CT and Dfg16CT confirmed the expression of both proteins (unpublished data). (B) Effect of Rim21 C-tail overexpression on Rim101 processing. OVY24 (wild type [WT] RIM101-HA) and OVY43 (rim21∆ RIM101-HA) were transformed with pADH1-Flag-Rim21CT (+) or empty vector (–). After a shift to pH 7.5, protein extracts were prepared and immunoblotted with anti-HA antibody. (C) Effect of ambient pH on Rim8 localization. OVY172 (RIM8-3GFP) was grown to mid log phase in YPD medium and then injected into a microfluidics device in SD medium containing 0.1 M HEPES, pH 4. Cells were then perfused with SD medium containing 0.1 M HEPES, pH 7, and examined by fluorescence and DIC microscopy before and after the change of medium at the indicated time. (D) Rim8 localization in cells overexpressing Rim21 C-tail. OVY172 (RIM8-3GFP) and OVY304 (rim21∆ RIM8-3GFP) were transformed with pADH1-Flag-Rim21CT or empty vector as indicated on the left. Cells were grown to mid log phase on SD medium (final pH 3.5) and examined by fluorescence and DIC microscopy. (E) Colocalization of Rim8 and overexpressed Rim21 C-tail. OVY172 (RIM8-3GFP) and OVY31(rim8∆) were transformed with pADH1-RFP-Rim21CT, grown to mid log phase on SD medium (final pH 3.5), and examined by fluorescence and DIC microscopy. (F) Effect of Rim21 C-tail overexpression on Rim8 phosphorylation. Y04414 was cotransformed with pHA-Rim8 (top) or a Rim8 truncated derivative (residues 1–460; bottom) and pADH1-Flag-Rim21CT (+) or empty vector (–). Transformants were grown to mid log phase (final pH 3.5), and protein extracts were prepared and immunoblotted with anti-HA antibody.
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Figure 3: Effect of Rim21 C-tail overexpression on RIM signaling and Rim8 localization and phosphorylation. (A) Phenotypic analysis of rim mutant strains overexpressing Rim21 or Dfg16 C-tails (CT). Y01150 (rim21∆), Y11806 (dfg16∆), Y06196 (rim9∆), and Y04414 (rim8∆) were transformed with pADH1-Flag-Rim21CT, pADH1-Flag-Dfg16CT, or empty vector (none). Growth tests in the presence of LiCl were as described for Figure 1D. Immunoblot analysis of transformants expressing Rim21CT and Dfg16CT confirmed the expression of both proteins (unpublished data). (B) Effect of Rim21 C-tail overexpression on Rim101 processing. OVY24 (wild type [WT] RIM101-HA) and OVY43 (rim21∆ RIM101-HA) were transformed with pADH1-Flag-Rim21CT (+) or empty vector (–). After a shift to pH 7.5, protein extracts were prepared and immunoblotted with anti-HA antibody. (C) Effect of ambient pH on Rim8 localization. OVY172 (RIM8-3GFP) was grown to mid log phase in YPD medium and then injected into a microfluidics device in SD medium containing 0.1 M HEPES, pH 4. Cells were then perfused with SD medium containing 0.1 M HEPES, pH 7, and examined by fluorescence and DIC microscopy before and after the change of medium at the indicated time. (D) Rim8 localization in cells overexpressing Rim21 C-tail. OVY172 (RIM8-3GFP) and OVY304 (rim21∆ RIM8-3GFP) were transformed with pADH1-Flag-Rim21CT or empty vector as indicated on the left. Cells were grown to mid log phase on SD medium (final pH 3.5) and examined by fluorescence and DIC microscopy. (E) Colocalization of Rim8 and overexpressed Rim21 C-tail. OVY172 (RIM8-3GFP) and OVY31(rim8∆) were transformed with pADH1-RFP-Rim21CT, grown to mid log phase on SD medium (final pH 3.5), and examined by fluorescence and DIC microscopy. (F) Effect of Rim21 C-tail overexpression on Rim8 phosphorylation. Y04414 was cotransformed with pHA-Rim8 (top) or a Rim8 truncated derivative (residues 1–460; bottom) and pADH1-Flag-Rim21CT (+) or empty vector (–). Transformants were grown to mid log phase (final pH 3.5), and protein extracts were prepared and immunoblotted with anti-HA antibody.
Mentions: Because Rim8 binding to Rim21 plays an essential role in RIM signaling (Figure 1D), we tested whether overexpression of the Rim21 C-tail fragment can bypass the requirement for the pH-sensing machinery in the activation of the RIM pathway. Lithium growth tests indicate that overexpression of Rim21 C-tail, but not Dfg16 C-tail, restores RIM signaling in rim21Δ, dfg16Δ, and rim9Δ mutants but not in a rim8Δ mutant (Figure 3A). Accordingly, Rim21 C-tail overexpression restores Rim101 processing in a rim21Δ mutant (Figure 3B). Thus overexpression of Rim21 C-tail can activate the RIM pathway in the absence of any component of the pH-sensing machinery but still in a Rim8-dependent manner.

Bottom Line: We show that Rim8 is phosphorylated in a pH-independent but Rim21-dependent manner by the plasma membrane-associated casein kinase 1 (CK1).Our results indicate that Rim8 phosphorylation prevents its accumulation at the plasma membrane at acidic pH and thereby inhibits RIM signaling.These findings support a model in which CK1-mediated phosphorylation of Rim8 contributes to setting a signaling threshold required to inhibit the RIM pathway at acidic pH.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, 28029 Madrid, Spain.

No MeSH data available.


Related in: MedlinePlus