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Eukaryotic protein kinases are enzymes that transfer a phosphoryl group (-PO<sub>3</sub><sup>2-</sup>) from adenosine triphosphate (or more rarely from adenosine diphosphate) to the hydroxyl group of serine, threonine, or tyrosine residue of a protein substrate. Phosphorylation of the substrate can affect its activity and/or conformation and, in turn, the physiogy of the cell. Protein kinases act as switches that turn on or off metabolic and signaling pathways, and they play central roles in development and responses to the environment. Also, unregulated versions of kinases that arise from tumor-promoting viruses promote cancer in humans.  The number of protein kinase genes (and the percentage of the genome) in bakers yeast<ref>PMID: 9020587</ref>, humans<ref> PMID:12471243</ref> and rice <ref>PMID:17172291</ref> are 113 (2%),518 (2%), and 1429 (5%), respectively. The catalytic domains of these enzymes occur alone or with other functional domains in a single polypetide chain. Protein kinases may be monomeric or multimeric or found in complexes with regulatory proteins.  
Eukaryotic protein kinases are enzymes that transfer a phosphoryl group (-PO<sub>3</sub><sup>2-</sup>) from adenosine triphosphate (or more rarely from adenosine diphosphate) to the hydroxyl group of serine, threonine, or tyrosine residue of a protein substrate. Phosphorylation of the substrate can affect its activity and/or conformation and, in turn, the physiogy of the cell. Protein kinases act as switches that turn on or off metabolic and signaling pathways, and they play central roles in development and responses to the environment. Also, unregulated versions of kinases that arise from tumor-promoting viruses promote cancer in humans.  The number of protein kinase genes (and the percentage of the genome) in bakers yeast<ref>PMID: 9020587</ref>, humans<ref> PMID:12471243</ref> and rice <ref>PMID:17172291</ref> are 113 (2%),518 (2%), and 1429 (5%), respectively. The catalytic domains of these enzymes occur alone or with other functional domains in a single polypetide chain. Protein kinases may be monomeric or multimeric or found in complexes with regulatory proteins.  


This article describes the general structure of protein kinase domains. It is based on the analysis of the primary structure of protein kinases by Hanks, Quinn, and Hunter <ref> PMID: 3291115</ref> in which the amino acid sequences of 65 protein kinases were aligned, and the revised analysis by Hanks and Hunter<ref> PMID: 7768349</ref>, and on the first three-dimensional structure of protein kinase to be published, that of protein kinase A (also called PKA or [[CAMP-dependent protein kinase]]) by Knighton et al.<ref> PMID: 1862342</ref> The results described in these papers apply to the basic structure of the great range of eukaryotic protein kinases known today.   
This article describes the general structure of protein kinase domains. It is based on the analysis of the primary structure of protein kinases by Hanks, Quinn, and Hunter<ref> PMID: 3291115</ref> in which the amino acid sequences of 65 protein kinases were aligned, and the revised analysis by Hanks and Hunter<ref> PMID: 7768349</ref>, and on the first three-dimensional structure of protein kinase to be published, that of protein kinase A (also called PKA or [[CAMP-dependent protein kinase]]) by Knighton et al.<ref> PMID: 1862342</ref> The results described in these papers apply to the basic structure of the great range of eukaryotic protein kinases known today.   


The crystal structure [[1atp]] contains the mouse PKA catalytic subunit (blue cartoon), inhibitor protein PKI (yellow cartoon), ATP (CPK wireframe), and two manganese ions (green spheres). The model at the left illustrates that catalytic domains of eukaryotic protein kinases have a small lobe and a large lobe (seen at the top and bottom of the model, respectively), and the catalytic cleft is located between them. The small lobe binds ATP and the large lobe binds the protein substrate modeled here by the inhibitor peptide, which has an alanine substituted for the serine in the phosphorylation motif RRxS. All of the molecular scenes include ATP, and some include the inhibitor peptide to illustrate kinase/substrate interactions.
The crystal structure [[1atp]] contains the mouse PKA catalytic subunit (blue cartoon), inhibitor protein PKI (yellow cartoon), ATP (CPK wireframe), and two manganese ions (green spheres). The model at the left illustrates that catalytic domains of eukaryotic protein kinases have a small lobe and a large lobe (seen at the top and bottom of the model, respectively), and the catalytic cleft is located between them. The small lobe binds ATP and the large lobe binds the protein substrate modeled here by the inhibitor peptide, which has an alanine substituted for the serine in the phosphorylation motif RRxS. All of the molecular scenes include ATP, and some include the inhibitor peptide to illustrate kinase/substrate interactions.

Revision as of 22:47, 21 August 2013

Eukaryotic Protein Kinase Catalytic DomainEukaryotic Protein Kinase Catalytic Domain

Eukaryotic protein kinases are enzymes that transfer a phosphoryl group (-PO32-) from adenosine triphosphate (or more rarely from adenosine diphosphate) to the hydroxyl group of serine, threonine, or tyrosine residue of a protein substrate. Phosphorylation of the substrate can affect its activity and/or conformation and, in turn, the physiogy of the cell. Protein kinases act as switches that turn on or off metabolic and signaling pathways, and they play central roles in development and responses to the environment. Also, unregulated versions of kinases that arise from tumor-promoting viruses promote cancer in humans. The number of protein kinase genes (and the percentage of the genome) in bakers yeast[1], humans[2] and rice [3] are 113 (2%),518 (2%), and 1429 (5%), respectively. The catalytic domains of these enzymes occur alone or with other functional domains in a single polypetide chain. Protein kinases may be monomeric or multimeric or found in complexes with regulatory proteins.

This article describes the general structure of protein kinase domains. It is based on the analysis of the primary structure of protein kinases by Hanks, Quinn, and Hunter[4] in which the amino acid sequences of 65 protein kinases were aligned, and the revised analysis by Hanks and Hunter[5], and on the first three-dimensional structure of protein kinase to be published, that of protein kinase A (also called PKA or CAMP-dependent protein kinase) by Knighton et al.[6] The results described in these papers apply to the basic structure of the great range of eukaryotic protein kinases known today.

The crystal structure 1atp contains the mouse PKA catalytic subunit (blue cartoon), inhibitor protein PKI (yellow cartoon), ATP (CPK wireframe), and two manganese ions (green spheres). The model at the left illustrates that catalytic domains of eukaryotic protein kinases have a small lobe and a large lobe (seen at the top and bottom of the model, respectively), and the catalytic cleft is located between them. The small lobe binds ATP and the large lobe binds the protein substrate modeled here by the inhibitor peptide, which has an alanine substituted for the serine in the phosphorylation motif RRxS. All of the molecular scenes include ATP, and some include the inhibitor peptide to illustrate kinase/substrate interactions.

Twelve Conserved SubdomainsTwelve Conserved Subdomains

Following is a tour of the twelve conserved subdomains (numbered starting at the amino terminal end of the catalytic domain) defined by Hanks and Hunter and using 1atp as a model.

1atp - Protein kinase A catalytic subunit in complex with ATP (wireframe), manganese, and inhibitor peptide PKI

Drag the structure with the mouse to rotate


contains two beta strands connected by the glycine-rich ATP-binding loop with the motif shown in ball and stick.

contains an that interacts with the phosphates of ATP.

is an alpha helix (helix C in bovine PKA) that connects to many parts of the kinase, and its orientation is critical for activity. In the active conformation of the kinase the (shown as blue ball and stick) in Subdomain III forms a salt bridge with the invariant lysine of Subdomain II (yellow ball and stick). This salt bridge couples subdomain III to ATP.

contains a beta strand and contributes to the core structure of the small lobe.

contains a hydrophobic beta strand in the small lobe and an alpha helix in the large lobe. The sequence that links these two secondary structures not only links together the small and large lobes of the kinase, but also contributes residues to the and also for . In PKA Glu 127 (blue ball and stick) interacts with both the ribose of ATP and the first Arg (yellow ball and stick) in the phosphorylation motif RRxS of a peptide substrate.

is a long alpha helix in the large lobe that parallels the alpha helix of subdomain IX.

contains the catalytic loop with the conserved motif HRDLKxxN (In PKA the H is a Y, instead). The is the catalytic base that accepts the hydrogen removed from the hydroxyl group being phosphorylated. Note the proximity of the glutamate residue to peptide residue that will be phosphorylated, here represented by an alanine (yellow ball and stick) in the inhibitor peptide. A substrate peptide would contain a serine instead of the alanine, and the hydroxyl group would narrow the gap between the substrate and the glutamate.

contains two beta strands link by the Mg-binding loop with the DFG motif. The chelates a Mg2+ ion (Mn2+ in the 1atp crystal structure) that bridges the gamma and beta phosphates of ATP and positions the gamma phosphate for transfer to the substrate.

contains several important features. The APE motif is located at the carboxyl end of this subdomain and the (blue ball and stick) in this motif forms a salt bridge with an arginine (yellow ball and stick) in in Subdomain XI. This salt bridge is critical for forming the stable kinase core and it provides an anchor for the movement of the activation loop (see below). In many protein kinases there is a phosphorylatable residue seven to ten residues upstream of the APE motif. In PKA it is a (blue ball and stick with the phosphate in CPK), which forms an ionic bond with the arginine (yellow ball and stick) in the YRDLKPEN motif of the catalytic loop and helps to position it for catalysis. Kinases that don't have a phosphorylatable residue in this loop often have an acididc residue that can form the salt bridge. Between the phosphorylated residue and the APE motif lies the (blue ball and stick), which interacts with the residue (yellow ball and stick) adjacent to the phosphorylated residue of the peptide substrate (yellow). The "P" residue is the one that is phosphoryated in the substrate, and the "P + 1" residue is the next residue in the sequence.

is a very hydrophobic alpha helix (helix F in mamallian PKA). It contains an invariant aspartate residue that is discussed below.

and contain three alpha helices (G, H, and I in mamallian PKA) that form the kinase core and which are involved in binding substrate proteins.

Beyond the Conserved SubdomainsBeyond the Conserved Subdomains

Functional structures that involve residues from more than one subdomain have been recognized by biochemical and molecular genetic studies coupled with three-dimensional structures of protein kinases.

The was first described by Taylor and Radzio-Andzelm[7]. It comprises amino acid residues between the DFG motif in subdomain VII to the APE motif in subdomain VIII. As it's name implies, it is involved in switching the activity of the kinase on and off. When the phosphorylatable residue in subdomain VIII (see above) is phosphorylated, the such that the active site cleft is accessible, the magnesium loop (DFG motif) and catalytic loop (HRDLKPxxN motif) are properly positioned for catalysis, and the P+1 loop can interact with the peptide substrate. The activation loop takes on a variety of conformations in inactive kinases[8], that disrupt one or all of these conformations.

Two hydrophobic (reviewed by Taylor and Kornev[9]) are important for the structure of active conformation of protein kinases. They are composed of amino acid residues that are non-contiguous in the primary structure. includes the adenine ring of ATP. In PKA it comprises residues (from top to bottom in the scene) A70, V57, ATP, L173, I174, L172, M128, M231, and L227, and it is directly anchored to amino end of helix F (Subdomain IX) contains residues L106, L95, F185, Y164, and it is anchored to helix F via a hydrogen bond between the invariant aspartate in helix F (yellow ball and stick) and the backbone nitrogen of Y164. This spine is assembled in the active conformation and disorganized in inactive conformations.

The residue[9] (chartreuse spacefill) is a part of subdomain V (blue) and it is located deep in the ATP-binding pocket (Subdomain I with its ATP binding loop are shown in yellow). The size of the gatekeeper residue determines the size of the binding pocket, and it is thus a gatekeeper for which nucleotides, ATP analogs, and inhibitors can bind[10]. In PKA and about 75% of all kinases it is a large residue, such as leucine, phenylalanine or methionine as seen here. In the remaining kinases, especially tyrosine kinases, the residue is larger, such as threonine or valine. The gatekeeper's location is [9] (gatekeeper is chartreuse, catalytic spine is blue, regulatory spine is orchid). Mutation of this residue in some kinases leads to activation of the kinase via enhanced autophosphorylation of the activation loop, and the unregulated kinase activity promotes cancer [11][12]. The gatekeeper's interaction with the two spines affects the orientation of the catalytic, magnesium binding, and activation loops.

ReferencesReferences

  1. ↑ Hunter T, Plowman GD. The protein kinases of budding yeast: six score and more. Trends Biochem Sci. 1997 Jan;22(1):18-22. PMID:9020587
  2. ↑ Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein kinase complement of the human genome. Science. 2002 Dec 6;298(5600):1912-34. PMID:12471243 doi:10.1126/science.1075762
  3. ↑ Dardick C, Chen J, Richter T, Ouyang S, Ronald P. The rice kinase database. A phylogenomic database for the rice kinome. Plant Physiol. 2007 Feb;143(2):579-86. Epub 2006 Dec 15. PMID:17172291 doi:10.1104/pp.106.087270
  4. ↑ Hanks SK, Quinn AM, Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42-52. PMID:3291115
  5. ↑ Hanks SK, Hunter T. Protein kinases 6. The eukaryotic protein kinase superfamily: kinase (catalytic) domain structure and classification. FASEB J. 1995 May;9(8):576-96. PMID:7768349
  6. ↑ Knighton DR, Zheng JH, Ten Eyck LF, Ashford VA, Xuong NH, Taylor SS, Sowadski JM. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):407-14. PMID:1862342
  7. ↑ Taylor SS, Radzio-Andzelm E. Three protein kinase structures define a common motif. Structure. 1994 May 15;2(5):345-55. PMID:8081750
  8. ↑ Huse M, Kuriyan J. The conformational plasticity of protein kinases. Cell. 2002 May 3;109(3):275-82. PMID:12015977
  9. ↑ 9.0 9.1 9.2 Taylor SS, Kornev AP. Protein kinases: evolution of dynamic regulatory proteins. Trends Biochem Sci. 2011 Feb;36(2):65-77. doi: 10.1016/j.tibs.2010.09.006. Epub, 2010 Oct 23. PMID:20971646 doi:10.1016/j.tibs.2010.09.006
  10. ↑ Zhang C, Kenski DM, Paulson JL, Bonshtien A, Sessa G, Cross JV, Templeton DJ, Shokat KM. A second-site suppressor strategy for chemical genetic analysis of diverse protein kinases. Nat Methods. 2005 Jun;2(6):435-41. PMID:15908922 doi:10.1038/nmeth764
  11. ↑ Emrick MA, Lee T, Starkey PJ, Mumby MC, Resing KA, Ahn NG. The gatekeeper residue controls autoactivation of ERK2 via a pathway of intramolecular connectivity. Proc Natl Acad Sci U S A. 2006 Nov 28;103(48):18101-6. Epub 2006 Nov 17. PMID:17114285 doi:10.1073/pnas.0608849103
  12. ↑ Azam M, Seeliger MA, Gray NS, Kuriyan J, Daley GQ. Activation of tyrosine kinases by mutation of the gatekeeper threonine. Nat Struct Mol Biol. 2008 Oct;15(10):1109-18. Epub 2008 Sep 14. PMID:18794843 doi:10.1038/nsmb.1486
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