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Eukaryotic Protein Kinase

Eukaryotic Protein kinases are enzymes that transfer a phosphoryl group (-PO₃^2-) 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, responses to the environment, and in diseases such as cancer.

In 1985 Hanks, Quinn, and Hunter provided the first analysis of protein kinase structure, based upon the alignment of amino acid sequences of 65 protein kinases. In 1995, Taylor et al, published the first x-ray crystal structure of a protein kinase, which was protein kinase A (PKA). The crystal structure and extensive biochemical work on PKA established it as a model for all eukaryotic protein kinases. The pioneering analysis of the primary structure of protein kinases (revised by Hanks and Hunter in 1995) and the three dimensional structure of PKA have proven to apply to the great range of protein kinases known today. Baker's yeast have ? protein kinase genes (?% of the genome),and humans and rice have ? (?%) and ? (?%), respectively.

Here, the twelve conserved subdomains defined by Hanks and Hunter are examined using PKA as the model protein. The crystal structure includes ATP.2Mg2+ and the inhibitor peptide that has an alanine substituted for serine in a substrate's phosphorylation motif RRxS. All of the scenes below include ATP, and some include the inhibitor peptide to illustrate kinase/substrate interactions.

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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 invariant 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.

links the small and large lobes. It 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 the peptide substrate.

is helix in the large lobe.

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. CHECK THIS FOR ACCURACY. 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.

, the Mg-binding loop with the DFG motif. The chelates a Mg²⁺ ion that bridges the gamma and beta phosphates of ATP and positions the gamma phosphate for transfer to the substrate.

the activation loop, the P+1 loop.

contains the APE motif. The glutamate in this motif forms a salt bridge with an arginine (yellow ball and stick) in in Subdomain X1. This salt bridge is critical for forming the stable kinase core. 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 HRDLKPEN motif of the catalytic loop and helps to position it for catalysis. Between the phosphorylated residue and the APE motif lies the which interacts with the residue adjacent to the phosphorylated residue of the peptide substrate.

is a very hydrophobic alpha helix (F in bovine PKA).

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

Two hydrophobic "spines" are important for the structure of active conformation of protein kinases (Taylor TIBS 2010). They are composed of amino acid residues that are non-contiguous in the primary structure.includes the adenine ring of ATP. In PKA the residues are 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.