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An autoinhibitory tyrosine motif in the cell-cycle-regulated Nek7 kinase is released through binding of Nek9.

Richards MW, O'Regan L, Mas-Droux C, Blot JM, Cheung J, Hoelder S, Fry AM, Bayliss R - Mol. Cell (2009)

Bottom Line: Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active.The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position.The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Research, London, UK.

ABSTRACT
Mitosis is controlled by multiple protein kinases, many of which are abnormally expressed in human cancers. Nek2, Nek6, Nek7, and Nek9 are NIMA-related kinases essential for proper mitotic progression. We determined the atomic structure of Nek7 and discovered an autoinhibited conformation that suggests a regulatory mechanism not previously described in kinases. Additionally, Nek2 adopts the same conformation when bound to a drug-like molecule. In both structures, a tyrosine side chain points into the active site, interacts with the activation loop, and blocks the alphaC helix. Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active. The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position. The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.

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Nek7 Encodes an Unusual NTE Motif that Is Required for Normal Kinase Activity(A) Sequence alignment of human Nek7, Nek6, and Nek2. Sequence conservation between Nek7 and Nek6 is highlighted in orange (identical), yellow (conservative substitution), and white (non-conserved). Residues that are identical in Nek2 are marked with asterisks. The ordered Nek7 NTE is marked with a gray box. Starting residues of Nek7 N-terminally truncated proteins are marked with black bars. Residues mutated in this study are marked with colored triangles: Tyr28Nek7 and Leu31Nek7, magenta; Asn33Nek7, black; Tyr97Nek7, cyan.(B) Stereo pair diagram depicts the unusual NTE motif in cartoon and sticks colored by conservation using the color scheme defined in (A). Residues mutated in this study are marked with colored triangles.(C) Sequence conservation between Nek7 and Nek6 mapped onto the surface of Nek7 using the color scheme defined in (A) in two views related by 180°.(D) In vitro kinase activity assay shows the relative activity of wild-type Nek7 (WT), the N-terminal truncations (Δ20, Δ30), and the NTE mutations N33D (ND) and Y28A/L31A (YA/LA). The upper panel shows the autoradiograph (32P) of a CB-stained gel in a single assay. The histograms in the lower panel show the average of three independent in vitro kinase assay experiments, and the error bars show the standard deviation.(E) Anti-His6 immunoblot of GST coprecipitation experiments using GST-tagged 9-CTD and GST control proteins to capture wild-type and mutant His6-tagged Nek7 proteins.
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fig6: Nek7 Encodes an Unusual NTE Motif that Is Required for Normal Kinase Activity(A) Sequence alignment of human Nek7, Nek6, and Nek2. Sequence conservation between Nek7 and Nek6 is highlighted in orange (identical), yellow (conservative substitution), and white (non-conserved). Residues that are identical in Nek2 are marked with asterisks. The ordered Nek7 NTE is marked with a gray box. Starting residues of Nek7 N-terminally truncated proteins are marked with black bars. Residues mutated in this study are marked with colored triangles: Tyr28Nek7 and Leu31Nek7, magenta; Asn33Nek7, black; Tyr97Nek7, cyan.(B) Stereo pair diagram depicts the unusual NTE motif in cartoon and sticks colored by conservation using the color scheme defined in (A). Residues mutated in this study are marked with colored triangles.(C) Sequence conservation between Nek7 and Nek6 mapped onto the surface of Nek7 using the color scheme defined in (A) in two views related by 180°.(D) In vitro kinase activity assay shows the relative activity of wild-type Nek7 (WT), the N-terminal truncations (Δ20, Δ30), and the NTE mutations N33D (ND) and Y28A/L31A (YA/LA). The upper panel shows the autoradiograph (32P) of a CB-stained gel in a single assay. The histograms in the lower panel show the average of three independent in vitro kinase assay experiments, and the error bars show the standard deviation.(E) Anti-His6 immunoblot of GST coprecipitation experiments using GST-tagged 9-CTD and GST control proteins to capture wild-type and mutant His6-tagged Nek7 proteins.

Mentions: Nek6 and Nek7 are 86% identical within their catalytic domains but are highly divergent in their N-terminal extensions (Figure 6A). The N-terminal extension to the catalytic domain of Nek7 (NTE, residues 20–33) adopts a highly unusual structure, an extended ordered loop wrapped around the side chain of Asn33 (Figure 6B). This side chain forms three H-bonds with the NTE backbone: the backbone oxygen of Gly27 and the backbone oxygen of Arg23 form H-bonds with the Asn33 side-chain nitrogen, and the backbone nitrogen of Arg23 forms an H-bond with the Asn33 side-chain oxygen. Substitution of Asn33 for any other amino acid would disrupt this network of H-bonds and destabilize the local structure. Notably, the equivalent position in Nek6 is an aspartic acid (Figures 6A and 6B). Mapping the sequence conservation between Nek6 and Nek7 onto the structure of Nek7 shows that the two kinases share an almost completely identical front face (Figure 6C, left), with most of the differences lying on the back face (Figure 6C, right). The large patch of nonconserved residues on the N lobe includes the ordered NTE of Nek7 and residues on the core catalytic domain that contact the NTE. The NTE of Nek7 sits on the core catalytic domain on a largely hydrophobic surface between β4 and the β2-β3 loop (Figure 6B). The differences between Nek6 and Nek7 in this region are hydrophilic for hydrophobic substitutions, suggesting that this region of Nek6 may be solvent exposed. For example the sequence “GVP” on β3 of Nek7 is replaced by “RKT” in Nek6, and Ala99Nek7 is replaced by aspartic acid. These changes, and the presence of aspartic acid at the equivalent position to Asn33Nek7, suggest that Nek6 does not have an NTE-like motif similar to that found in Nek7.


An autoinhibitory tyrosine motif in the cell-cycle-regulated Nek7 kinase is released through binding of Nek9.

Richards MW, O'Regan L, Mas-Droux C, Blot JM, Cheung J, Hoelder S, Fry AM, Bayliss R - Mol. Cell (2009)

Nek7 Encodes an Unusual NTE Motif that Is Required for Normal Kinase Activity(A) Sequence alignment of human Nek7, Nek6, and Nek2. Sequence conservation between Nek7 and Nek6 is highlighted in orange (identical), yellow (conservative substitution), and white (non-conserved). Residues that are identical in Nek2 are marked with asterisks. The ordered Nek7 NTE is marked with a gray box. Starting residues of Nek7 N-terminally truncated proteins are marked with black bars. Residues mutated in this study are marked with colored triangles: Tyr28Nek7 and Leu31Nek7, magenta; Asn33Nek7, black; Tyr97Nek7, cyan.(B) Stereo pair diagram depicts the unusual NTE motif in cartoon and sticks colored by conservation using the color scheme defined in (A). Residues mutated in this study are marked with colored triangles.(C) Sequence conservation between Nek7 and Nek6 mapped onto the surface of Nek7 using the color scheme defined in (A) in two views related by 180°.(D) In vitro kinase activity assay shows the relative activity of wild-type Nek7 (WT), the N-terminal truncations (Δ20, Δ30), and the NTE mutations N33D (ND) and Y28A/L31A (YA/LA). The upper panel shows the autoradiograph (32P) of a CB-stained gel in a single assay. The histograms in the lower panel show the average of three independent in vitro kinase assay experiments, and the error bars show the standard deviation.(E) Anti-His6 immunoblot of GST coprecipitation experiments using GST-tagged 9-CTD and GST control proteins to capture wild-type and mutant His6-tagged Nek7 proteins.
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fig6: Nek7 Encodes an Unusual NTE Motif that Is Required for Normal Kinase Activity(A) Sequence alignment of human Nek7, Nek6, and Nek2. Sequence conservation between Nek7 and Nek6 is highlighted in orange (identical), yellow (conservative substitution), and white (non-conserved). Residues that are identical in Nek2 are marked with asterisks. The ordered Nek7 NTE is marked with a gray box. Starting residues of Nek7 N-terminally truncated proteins are marked with black bars. Residues mutated in this study are marked with colored triangles: Tyr28Nek7 and Leu31Nek7, magenta; Asn33Nek7, black; Tyr97Nek7, cyan.(B) Stereo pair diagram depicts the unusual NTE motif in cartoon and sticks colored by conservation using the color scheme defined in (A). Residues mutated in this study are marked with colored triangles.(C) Sequence conservation between Nek7 and Nek6 mapped onto the surface of Nek7 using the color scheme defined in (A) in two views related by 180°.(D) In vitro kinase activity assay shows the relative activity of wild-type Nek7 (WT), the N-terminal truncations (Δ20, Δ30), and the NTE mutations N33D (ND) and Y28A/L31A (YA/LA). The upper panel shows the autoradiograph (32P) of a CB-stained gel in a single assay. The histograms in the lower panel show the average of three independent in vitro kinase assay experiments, and the error bars show the standard deviation.(E) Anti-His6 immunoblot of GST coprecipitation experiments using GST-tagged 9-CTD and GST control proteins to capture wild-type and mutant His6-tagged Nek7 proteins.
Mentions: Nek6 and Nek7 are 86% identical within their catalytic domains but are highly divergent in their N-terminal extensions (Figure 6A). The N-terminal extension to the catalytic domain of Nek7 (NTE, residues 20–33) adopts a highly unusual structure, an extended ordered loop wrapped around the side chain of Asn33 (Figure 6B). This side chain forms three H-bonds with the NTE backbone: the backbone oxygen of Gly27 and the backbone oxygen of Arg23 form H-bonds with the Asn33 side-chain nitrogen, and the backbone nitrogen of Arg23 forms an H-bond with the Asn33 side-chain oxygen. Substitution of Asn33 for any other amino acid would disrupt this network of H-bonds and destabilize the local structure. Notably, the equivalent position in Nek6 is an aspartic acid (Figures 6A and 6B). Mapping the sequence conservation between Nek6 and Nek7 onto the structure of Nek7 shows that the two kinases share an almost completely identical front face (Figure 6C, left), with most of the differences lying on the back face (Figure 6C, right). The large patch of nonconserved residues on the N lobe includes the ordered NTE of Nek7 and residues on the core catalytic domain that contact the NTE. The NTE of Nek7 sits on the core catalytic domain on a largely hydrophobic surface between β4 and the β2-β3 loop (Figure 6B). The differences between Nek6 and Nek7 in this region are hydrophilic for hydrophobic substitutions, suggesting that this region of Nek6 may be solvent exposed. For example the sequence “GVP” on β3 of Nek7 is replaced by “RKT” in Nek6, and Ala99Nek7 is replaced by aspartic acid. These changes, and the presence of aspartic acid at the equivalent position to Asn33Nek7, suggest that Nek6 does not have an NTE-like motif similar to that found in Nek7.

Bottom Line: Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active.The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position.The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cancer Research, London, UK.

ABSTRACT
Mitosis is controlled by multiple protein kinases, many of which are abnormally expressed in human cancers. Nek2, Nek6, Nek7, and Nek9 are NIMA-related kinases essential for proper mitotic progression. We determined the atomic structure of Nek7 and discovered an autoinhibited conformation that suggests a regulatory mechanism not previously described in kinases. Additionally, Nek2 adopts the same conformation when bound to a drug-like molecule. In both structures, a tyrosine side chain points into the active site, interacts with the activation loop, and blocks the alphaC helix. Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active. The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position. The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.

Show MeSH
Related in: MedlinePlus