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Eukaryotic Protein Kinase Catalytic Domain

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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 first section of this article relates the twelve conserved subdomains recognized in the primary structures of protein kinase catalytic domains[4][5] to the three-dimensional structure of protein kinase A (also called PKA or CAMP-dependent protein kinase)[6][7]. The results described in these classic papers apply to the basic structure of the great range of eukaryotic protein kinases known today.

The second section of this article examines functional structures and assemblies of protein kinase catalytic domains and compares active and inactive conformations.

Tour of Structural Features

Active and inactive structures

The kinase structure used in the above tour is that of the active conformation of PKA. While active conformations of protein kinases are very similar, there is great variation in the inactive conformations of protein kinases, but all involve misalignment of one or more of the structures, subdomain III (C-helix in PKA) and the catalytic, magnesium binding, and activation loops[10].

To get an idea of the structural differences that occur during a catalytic cycle and in active and inactive enzymes, use the links below to compare inactive, unphosphorylated PKA 4dfy (activation loop threonine is not phosphorylated), active apo PKA 1j3h, and active PKA in complex with ANP and PKI 1atp (the same structure used above), shown in the left, middle, and right frames, respectively. 4dfy shows the structure of an inactive form of PKA, in which the internal structure is disorganized due to the lack of phosphorylation of threonine 197 in the activation loop. Phosphorylation of this residue is required for formation of hydrogen bonds that are critical for alignment of structures to form the active site. 1j3h and 1atp show the open and closed structures assumed by PKA during the catalytic cycle. Note that some residues in 1j3h and 4dfy are not depicted in the models, because they are disordered and not resolved in the structures.

Click on all three links with same number to compare the indicated features. Legends for each set of scenes are below. To reset the structures, reload the page.

4dfy - apo unphosphorylated PKA, inactive


4dfy


1j3h - apo PKA, open conformation


1j3h


1atp - PKA with ANP and PKI; closed and active


1atp


Scene legends
1. In these scenes the catalytic domains are shown in spacefill, with the large lobe in silver and the small lobe in blue. To aid viewing, The N and C terminal sequences are in cartoon. Stop the rotation and use your mouse to get a good look at the catalytic cleft, which in 1ATP is closed around ANP. Two sets of residues are shown in yellow and red, respectively, to show the degree to which the cleft opens, and the two lobes twist with respect to each other. The yellow residues are Gly52 from the GxGxxG motif and Thr 201 in the activation loop. The red residues are His 87 in subdomain III (the C helix) and phosphorthreonine 197 in the activation loop. (The activation loop of the unphosphorylated PKA is disordered, and thus not represented in the crystal structure.) Note the difference in distance and alignment of these pairs of residues. The small lobe is rotated 18° relative to the active conformation. In the closed, active conformation His 87 and phosphoThr 197 have an ionic interaction, whereas in the open conformation they are too far away from each other to interact.

2. These scenes show the catalytic spine in blue space fill and the regulatory spine in orchid spacefill. The spines are assembled in the closed and open active kinases (left and middle scenes), but disorganized in the inactive kinase (right).

3. These scenes show the alignments of structures critical for activity. The yellow Cα-trace is the DFG-activation loop sequence, the blue trace is the catalytic loop, and the orchid trace is the C-helix (subdomain III). In ball and stick are residues critical for catalytic activity: yellow is the D in DFG, which binds Mg2+; blue is the D in the YRDKLPEN, which is the catalytic base; cyan is the invariant K of subdomain II, which binds the phosphates of ATP; and orchid is the invariant E of subdomain III. The positions needed for catalysis can be seen in the closed, active kinase (left). The two D's and K are pointing toward ANP, and the E is bound to the K. The latter pulls the C-helix into position. In the open structure (middle) the elements of the large lobe are in place but the K of the small lobe is far away from the ANP binding site. Upon ATP binding the K interacts with the phosphates and the two lobes close. The view of the inactive structure (right) is oriented so that the backbones of the catalytic loop (blue) and ends of the activation loop (yellow) are positioned like those in the other two structures. The other residues of the activation loop are not shown because they were not resolved in the crystal structure because of their flexibility. The side chain of the D in the catalytic loop (blue ball and stick)points away from the ATP binding pocket, and the C-helix is rotated upward. The assembly of these elements depends on the phosphorylation of threonine 197 in the activation loop. The phosphate of the residue forms five critical bonds that align the active site structures[14].

Regulation of Protein Kinase Activity

There are a variety of ways that the activity of protein kinases are regulated. Here are a few examples. Some are regulated via phosphorylation of residue(s) in the activation loop by either an upstream protein kinase (such as mitogen-activated protein kinase phosphorylation by MAPKK) or by autophosphosphorylation stimulated by the binding of a ligand (such as the insulin receptor kinase[15]). Others are activated by binding with other proteins, which brings the kinase into the active conformation. The PKA C subunit, having been constitutively phosphorylated by an upstream kinase, is active when released from a complex with the regulatory subunit upon the binding of cAMP (see cAMP-dependent protein kinase). Calcium-dependent protein kinase has calcium-binding domain that blocks the active site in the absence of calcium[16]. Upon binding calcium the latter domain undergoes a dramatic conformational change and it moves to a binding site that is on opposite side of the kinase, thus unblocking the catalytic cleft.

References

  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. 5.0 5.1 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. 7.0 7.1 Knighton DR, Zheng JH, Ten Eyck LF, Xuong NH, Taylor SS, Sowadski JM. Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):414-20. PMID:1862343
  8. Taylor SS, Radzio-Andzelm E. Three protein kinase structures define a common motif. Structure. 1994 May 15;2(5):345-55. PMID:8081750
  9. Huse M, Kuriyan J. The conformational plasticity of protein kinases. Cell. 2002 May 3;109(3):275-82. PMID:12015977
  10. 10.0 10.1 10.2 10.3 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
  11. 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
  12. 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
  13. 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
  14. Steichen JM, Kuchinskas M, Keshwani MM, Yang J, Adams JA, Taylor SS. Structural basis for the regulation of protein kinase A by activation loop phosphorylation. J Biol Chem. 2012 Feb 10. PMID:22334660 doi:10.1074/jbc.M111.335091
  15. Hubbard SR, Wei L, Ellis L, Hendrickson WA. Crystal structure of the tyrosine kinase domain of the human insulin receptor. Nature. 1994 Dec 22-29;372(6508):746-54. PMID:7997262 doi:http://dx.doi.org/10.1038/372746a0
  16. Wernimont AK, Artz JD, Finerty P Jr, Lin YH, Amani M, Allali-Hassani A, Senisterra G, Vedadi M, Tempel W, Mackenzie F, Chau I, Lourido S, Sibley LD, Hui R. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium. Nat Struct Mol Biol. 2010 May;17(5):596-601. Epub 2010 May 2. PMID:20436473 doi:10.1038/nsmb.1795

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Alice Harmon

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