Protein Kinase Substrate Recognition

The reversible addition of phosphate groups to proteins is important for the transmission of signals within eukaryotic cells and, as a result, protein phosphorylation and dephosphorylation regulate many diverse cellular processes. As the number of known protein kinases has increased at an ever-accelerating pace, it has become more challenging to determine which protein kinases interact with which substrates in the cell. The determination of consensus phosphorylation site motifs by amino acid sequence alignment of known substrates has proven useful in this pursuit. These motifs can be helpful for predicting phosphorylation sites for specific protein kinases within a potential protein substrate.

Since the determinants of protein kinase specificity involve complex 3-dimensional interactions, these motifs, short amino-acid sequences describing the primary structure around the phosphoacceptor residue, are a significant oversimplification of the issue (see (1) for review). They do not take into account possible secondary and tertiary structural elements, or determinants from other polypeptide chains or from distant locations within the same chain. Furthermore, not all of the residues described in a particular specificity motif may carry the same weight in determining recognition and phosphorylation by the kinase. As a consequence, they should be used with some caution.

On the other hand, many of the residues within these consensus sequences have in fact proven to be crucial recognition elements, and the very simplicity of these motifs has made them useful in the study of protein kinases and their substrates. In addition to the prediction of phosphorylation sites, short synthetic oligopeptides based on consensus motifs are often excellent substrates for protein kinase activity assays.

The table below summarizes some of the known data about specificity motifs for various well-studied protein kinases, along with examples of known phosphorylation sites in specific proteins (see (2) for a more extensive list). Phosphoacceptor residue is indicated in red, amino acids which can function interchangeably at a particular residue are separated by a slash (/), and residues which do not appear to contribute strongly to recognition are indicated by an “X”.

Some protein kinases such as CKI and GSK-3 contain phosphoamino acid residues in their recognition motifs, and have been termed “hierarchical” protein kinases (see (3) for review). They often require prior phosphorylation by another kinase at a residue in the vicinity of their own phosphorylation site. S(P) represents such preexisting phosphoserine residues.

Protein Kinase	Recognition Motifs^a	Phosphorylation Sites^b	Protein Substrate (reference)
cAMP-dependent Protein Kinase (PKA, cAPK)	R-X-S/T^c R-R/K-X-S/T	Y₇LRRASLAQLT F₁RRLSIST A₂₉GARRKASGPP	pyruvate kinase (2) phosphorylase kinase, a chain (2) histone H1, bovine (2)
Casein Kinase I (CKI, CK-1)	S(P)-X-X-S/T	R₄TLS(P)VSSLPGL D₄₃IGS(P)ES(P)TEDQ	glycogen synthase, rabbit muscle (4) a_s1-casein (4)
Casein Kinase II (CKII, CK-2)	S/T-X-X-E	A₇₂DSESEDEED L₃₇ESEEEGVPST E₂₆DNSEDEISNL	PKA regulatory subunit, R_II (2) p34^cdc2, human (5) acetyl-CoA carboxylase (2)
Glycogen Synthase Kinase 3 (GSK-3)	S-X-X-X-S(P)	S₆₄₁VPPSPSLS(P) S₆₄₁VPPS(P)PSLS(P)	glycogen synthase, human (site 3b) (6,2) glycogen synthase, human (site 3a) (6,2)
Cdc2 Protein Kinase; CDK2-cyclin A	S/T-P-X-R/K^c	P₁₃AKTPVK H₁₂₂STPPKKKRK	histone H1, calf thymus (2) large T antigen (2)
Calmodulin-dependent Protein Kinase II (CaMK II)	R-X-X-S/T R-X-X-S/T-V	N₂YLRRRLSDSN K₁₉₁MARVFSVLR	synapsin (site 1) (2) calcineurin (2)
Mitogen-activated Protein Kinase (Extracellular Signal-regulated Kinase) (MAPK, Erk)	P-X-S/T-P^d X-X-S/T-P	P₂₄₄LSP P₉₂SSP V₄₂₀LSP	c-Jun (7) cyclin B (7) Elk-1 (7)
Abl Tyrosine Kinase	I/V/L-Y-X-X-P/F^e

Single-letter Amino Acid Code:
A = alanine, C = cysteine, D = aspartic acid, E = glutamic acid, F = phenylalanine, G = glycine, H = histidine, I = isoleucine, K = lysine, L = leucine, M = methionine, N = asparagine, P = proline, Q = glutamine, R = arginine, S = serine, T = threonine, W = tryptophan, V = valine, Y = tyrosine, X = any amino acid

^a	Recognition motifs are taken from (2) except where noted. Consult this reference for a comprehensive list of phosphorylation site sequences and specificity motifs.
^b	Subscripted numbers refer to the position of the first residue within the given polypeptide chain.
^c	From (1). See refs (1) and (10) for discussion of substrate recognition by Cdc2 Protein Kinase and CDK2-cyclin A.
^d	From (7).
^e	From (8). See refs (8) and (9) for discussion of substrate recognition by Abl.

References:

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