Phosphorylation-dependent PIH1D1 interactions define substrate specificity of the R2TP cochaperone complex

Zuzana Hořejší; Lasse Stach; Thomas G Flower; Dhira Joshi; Helen Flynn; J Mark Skehel; Nicola J O'Reilly; Roksana W Ogrodowicz; Stephen J Smerdon; Simon J Boulton

doi:10.1016/j.celrep.2014.03.013

Phosphorylation-dependent PIH1D1 interactions define substrate specificity of the R2TP cochaperone complex

Zuzana Hořejší, Lasse Stach, Thomas G Flower, Dhira Joshi, Helen Flynn, J Mark Skehel, Nicola J O'Reilly, Roksana W Ogrodowicz, Stephen J Smerdon, Simon J Boulton

Cancer and Genomic Sciences

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Abstract

The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit.

Original language	English
Pages (from-to)	19-26
Number of pages	8
Journal	Cell Reports
Volume	7
Issue number	1
Early online date	20 Mar 2014
DOIs	https://doi.org/10.1016/j.celrep.2014.03.013
Publication status	Published - 10 Apr 2014

Keywords

Amino Acid Sequence
Apoptosis Regulatory Proteins/chemistry
Cell Line, Tumor
Crystallography, X-Ray/methods
HEK293 Cells
HSP90 Heat-Shock Proteins/chemistry
Humans
Models, Molecular
Molecular Chaperones/genetics
Molecular Sequence Data
Multiprotein Complexes/chemistry
Phosphorylation
Protein Structure, Tertiary
Proto-Oncogene Proteins c-ets/chemistry
Sequence Homology, Amino Acid
Substrate Specificity

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10.1016/j.celrep.2014.03.013Licence: Creative Commons: Attribution-NonCommercial-NoDerivs (CC BY-NC-ND)

HořejšíZ2014PhosphorylationFinal published version, 2.17 MBLicence: Creative Commons: Attribution-NonCommercial-NoDerivs (CC BY-NC-ND)

https://doi.org/10.1016/j.celrep.2014.03.013Licence: Creative Commons: Attribution-NonCommercial-NoDerivs (CC BY-NC-ND)

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title = "Phosphorylation-dependent PIH1D1 interactions define substrate specificity of the R2TP cochaperone complex",

abstract = "The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit. ",

keywords = "Amino Acid Sequence, Apoptosis Regulatory Proteins/chemistry, Cell Line, Tumor, Crystallography, X-Ray/methods, HEK293 Cells, HSP90 Heat-Shock Proteins/chemistry, Humans, Models, Molecular, Molecular Chaperones/genetics, Molecular Sequence Data, Multiprotein Complexes/chemistry, Phosphorylation, Protein Structure, Tertiary, Proto-Oncogene Proteins c-ets/chemistry, Sequence Homology, Amino Acid, Substrate Specificity",

author = "Zuzana Ho{\v r}ej{\v s}{\'i} and Lasse Stach and Flower, {Thomas G} and Dhira Joshi and Helen Flynn and Skehel, {J Mark} and O'Reilly, {Nicola J} and Ogrodowicz, {Roksana W} and Smerdon, {Stephen J} and Boulton, {Simon J}",

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language = "English",

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T1 - Phosphorylation-dependent PIH1D1 interactions define substrate specificity of the R2TP cochaperone complex

AU - Hořejší, Zuzana

AU - Stach, Lasse

AU - Flower, Thomas G

AU - Joshi, Dhira

AU - Flynn, Helen

AU - Skehel, J Mark

AU - O'Reilly, Nicola J

AU - Ogrodowicz, Roksana W

AU - Smerdon, Stephen J

AU - Boulton, Simon J

PY - 2014/4/10

Y1 - 2014/4/10

N2 - The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit.

AB - The R2TP cochaperone complex plays a critical role in the assembly of multisubunit machines, including small nucleolar ribonucleoproteins (snoRNPs), RNA polymerase II, and the mTORC1 and SMG1 kinase complexes, but the molecular basis of substrate recognition remains unclear. Here, we describe a phosphopeptide binding domain (PIH-N) in the PIH1D1 subunit of the R2TP complex that preferentially binds to highly acidic phosphorylated proteins. A cocrystal structure of a PIH-N domain/TEL2 phosphopeptide complex reveals a highly specific phosphopeptide recognition mechanism in which Lys57 and 64 in PIH1D1, along with a conserved DpSDD phosphopeptide motif within TEL2, are essential and sufficient for binding. Proteomic analysis of PIH1D1 interactors identified R2TP complex substrates that are recruited by the PIH-N domain in a sequence-specific and phosphorylation-dependent manner suggestive of a common mechanism of substrate recognition. We propose that protein complexes assembled by the R2TP complex are defined by phosphorylation of a specific motif and recognition by the PIH1D1 subunit.

KW - Amino Acid Sequence

KW - Apoptosis Regulatory Proteins/chemistry

KW - Cell Line, Tumor

KW - Crystallography, X-Ray/methods

KW - HEK293 Cells

KW - HSP90 Heat-Shock Proteins/chemistry

KW - Humans

KW - Models, Molecular

KW - Molecular Chaperones/genetics

KW - Molecular Sequence Data

KW - Multiprotein Complexes/chemistry

KW - Phosphorylation

KW - Protein Structure, Tertiary

KW - Proto-Oncogene Proteins c-ets/chemistry

KW - Sequence Homology, Amino Acid

KW - Substrate Specificity

U2 - 10.1016/j.celrep.2014.03.013

DO - 10.1016/j.celrep.2014.03.013

M3 - Article

C2 - 24656813

SN - 2211-1247

VL - 7

SP - 19

EP - 26

JO - Cell Reports

JF - Cell Reports

IS - 1

ER -

Phosphorylation-dependent PIH1D1 interactions define substrate specificity of the R2TP cochaperone complex

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Keywords

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