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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 Motifsa |
Phosphorylation Sitesb |
Protein Substrate (reference) |
| cAMP-dependent Protein Kinase
(PKA, cAPK) |
R-X-S/Tc
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)
as1-casein (4) |
| Casein Kinase II (CKII, CK-2) |
S/T-X-X-E |
A72DSESEDEED
L37ESEEEGVPST
E26DNSEDEISNL |
PKA regulatory subunit, RII
(2)
p34cdc2, 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/Kc |
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-Pd
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/Fe |
|
|
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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. |
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References:
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E. G. (1991) J. Biol. Chem. 266, 15555–15558.
- Pearson, R. B., and Kemp, B.
E. (1991). In T. Hunter and B. M. Sefton (Eds.), Methods in Enzymology
Vol. 200, (pp. 62–81). San Diego: Academic Press.
- Roach, P. J. (1991) J. Biol.
Chem. 266, 14139–14142.
- Flotow, H. et al. (1990) J.
Biol. Chem. 265, 14264–14269.
- Russo, G. L. et al. (1992) J.
Biol. Chem. 267, 20317–20325.
- Fiol, C. J. et al. (1990) J.
Biol. Chem. 265, 6061–6065.
- Davis, R. J. (1993) J. Biol.
Chem. 268, 14553–14556.
- Songyang, Z. et al. (1995) Nature
373, 536–539.
- Geahlen, R. L. and Harrison,
M. L. (1990). In B. E. Kemp (Ed.), Peptides and Protein Phosphorylation,
(pp. 239–253). Boca Raton: CRC Press.
- Stevenson, L.M. et al. (2003) J. Biol. Chem. 278, 50956–50960.
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