Download Distinct Strategies in Ser/Thr and Tyr

doi:10.1016/j.jmb.2004.04.043
J. Mol. Biol. (2004) 339, 1025–1039
REVIEW
Structural Modes of Stabilization of Permissive
Phosphorylation Sites in Protein Kinases: Distinct
Strategies in Ser/Thr and Tyr Kinases
A. Krupa1, G. Preethi2 and N. Srinivasan1*
1
Molecular Biophysics Unit
Indian Institute of Science
Bangalore 560012, India
2
Department of Biotechnology
Bharathiar University
Coimbatore 641043, India
Protein kinases phosphorylate several cellular proteins providing control
mechanisms for various signalling processes. Their activity is impeded in
a number of ways and restored by alteration in their structural properties
leading to a catalytically active state. Most protein kinases are subjected
to positive and negative regulation by phosphorylation of Ser/Thr/Tyr
residues at specific sites within and outside the catalytic core.
The current review describes the analysis on 3D structures of protein
kinases that revealed features distinct to active states of Ser/Thr and Tyr
kinases. The nature and extent of interactions among well-conserved residues surrounding the permissive phosphorylation sites differ among the
two classes of enzymes. The network of interactions of highly conserved
Arg preceding the catalytic base that mediates stabilization of the activation segment exemplifies such diverse interactions in the two groups
of kinases.
The N-terminal and the C-terminal lobes of various groups of protein
kinases further show variations in their extent of coupling as suggested
from the extent of interactions between key functional residues in activation segment and the N-terminal aC-helix. We observe higher similarity
in the conformations of ATP bound to active forms of protein kinases
compared to ATP conformations in the inactive forms of kinases.
The extent of structural variations accompanying phosphorylation of
protein kinases is widely varied. The comparison of their crystal structures and the distinct features observed are hoped to aid in the understanding of mechanisms underlying the control of the catalytic activity of
distinct subgroups of protein kinases.
q 2004 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: protein kinase; phosphorylation; regulation; stabilization of
active site; active and inactive state
Introduction
Phosphorylation of intra-cellular proteins by
protein kinases serve as molecular switches of
various cellular processes including metabolism,
gene expression, cell division, motility and
differentiation.1 – 3 Toggling between distinct actiAbbreviations used: PKA, protein kinase A; PKB,
protein kinase B; GS, glycine-serine; GSK-3, glycogen
synthase kinase-3; GSK, glycogen synthase kinase; CK2,
casein kinase-2; CSK, C-terminal Src kinase; HCK,
hemapoietic cell kinase.
E-mail address of the corresponding author:
[email protected]
vation states endowed with unique conformational
features brings about the tight regulation of these
enzymes.4 Multiple modes of regulation of protein
kinases have enabled perception of specific signals
leading to selective activation of downstream
signalling cascades.5,6
Critical structural features of catalytically active
states of all protein kinases carrying out phospho
transfer are very similar.4,7 Phosphorylation on the
activation segment, a key regulatory element, acts
as a switch for the catalytic activity of protein
kinases belonging to distinct subgroups of Ser/
Thr and Tyr kinase families.8 Protein kinases regulated by such phosphorylation form a network
of residue contacts at the active site that are well
0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
1026
Review: Phosphorylation Sites in Kinases
Figure 1. The 3D fold of catalytic
core of Ser/Thr or Tyr kinase with
their key functional elements is
shown. This Figure as well as
Figures 3, 4, 6, 7, 9, and 11, are
generated using SETOR.37
conserved in their “on” or maximally active state.
The key residues involved in stabilization of the
catalytic site as described above are invariant
across different subgroups of kinases with similar
mechanism of regulation.5 The focus of this review
is to provide our current understanding on the
extent of conservation of the nature of residue contacts in kinases at the activation loop as revealed
from the survey of available 3D structures in active
and inactive states. We describe the variations in
the residue contacts observed across Ser/Thr and
Tyr kinase families. Further, the extension of these
contacts to residues conserved specifically in Ser/
Thr and Tyr kinases and their implications in alternate modes of stabilization of activation segment
are discussed. Apart from the positive regulatory
phosphorylation sites within the catalytic core, the
inhibitory phosphorylation sites and their residue
environment have been surveyed to explore the
conservation of their interaction network. The
varying residue environment of these phosphosites as exemplified by cyclin dependent kinases
provides insight into their mechanism of inhibition. Analysis of other critical functional features
including the Lys-Glu salt-bridge dyad and nucleotide binding of the protein kinases in active and
inactive states, reveal differences that provide a
platform for further investigations on their role in
catalysis and ligand binding.
The 3D structure of kinase catalytic core
The first 3D structure of a protein kinase, protein
kinase A9 (PKA) as determined by X-ray crystallography revealed the basic bilobal scaffold that
has now been observed in all the protein kinase
structures (Figure 1) solved to date. The N-terminal
lobe of the kinase fold comprises of an anti-parallel
b-sheet made of five b-strands and a single a-helix
referred to as aC-helix. The C-terminal lobe is
larger and is mainly composed of a-helices. The
nucleotide binding and the substrate-binding
pocket are located in the cleft between the two
lobes. The phosphate groups of ATP are positioned
for phospho transfer by their interactions with conserved residues in the N and C-terminal lobes.
These include a glycine-rich loop characterized by
“GXGXXG” (where X represents any amino acid)
motif between the b1 and b2 strands, a Lys residue
localized by a salt bridge formed by a Lys-Glu pair
(K72† and E91) and Mg2þ ions. Conserved Asn
(N171) and Asp (D184) further coordinate the
metal ions. The catalytic loop situated in the
C-terminal lobe contains the aspartate (D166)
referred to as catalytic base that facilitates extraction
of proton from the hydroxyl side-chains of phospho-sites of the substrates. The activation segment
of 20–30 residues in length caps the C-terminal
lobe. This segment forms a part of the substratebinding pocket and shows high structural variation
in the active and inactive kinase structures.
Phosphorylation of the activation segment of
protein kinases
The common catalytic scaffold shared by the
eukaryotic protein kinases switches between two
extreme conformations of on and off states of
high and least activity, respectively, in a number
† Unless otherwise specified residue numbering as in
the crystal structure of PKA9 is followed.
Review: Phosphorylation Sites in Kinases
of ways. The transitions between two distinct functional states are mediated by a variety of strategies
in different kinases such as phosphorylation,
and interactions of additional domains within
the protein kinases and/or by binding to other
regulatory proteins.4
Phosphorylation of the activation segment is a
conserved feature in the regulation of most protein
kinases belonging to the group of “RD” kinases.5
The RD kinases contain an Arg preceding the catalytic aspartate in the catalytic loop. Phosphorylation at the conserved Ser/Thr in the activation
segment serves as a nucleation centre, neutralizing
a cluster of positively charged residues including
Arg of the RD motif.5 Such electrostatic interactions
are crucial for the relative orientation of the “DFG”
motif of the activation segment and the other catalytic and substrate binding residues to facilitate
phosphotransfer. The conformation of the activation segment is controlled by the phosphorylation, which promotes secondary changes
including re-orientation between the lobes, relief
of autoinhibition and the correct disposition of
catalytic residues. The basic residues that interact
with the phospho-amino acid on the activation segment are highly conserved across various kinases
and are referred to as contact residues (Figures
2(a) and (b) and 3) in the subsequent discussion.
Protein kinases in the off state adopt distinct
conformation characteristic to their inherent mode
of regulation. The nature of interactions between
phospho-amino acid and the contact residues
observed for various protein kinases in their active
and inactive states has been described earlier.5
These interactions are well conserved in the on
state of most protein kinases consistent with the
notion of a unique conformation and functional
features of the protein kinases in their active states.
Crystal structures of protein kinases in on state
also reveal distinct pattern of interaction confined
to either Ser/Thr kinases or tyrosine kinases.
These differences at the active site are discussed
below.
Distinct strategies of stabilization of the
activation segment
The crystal structures of protein kinases of the
RD group requiring phosphorylation at the activation segment and available in both active and
inactive states are listed in Table 1. Comparison of
the 3D structures of Ser/Thr and tyrosine kinases
in on state, reveals a striking difference in the
nature of contacts of the phospho-amino acid in
the activation segment with the highly conserved
Arg of the RD motif. Ion pair between the Arg
residue (in RD motif) and the phospho-amino acid
that forms a network of electrostatic interactions
with other contact residues (Figures 2(a) and 3),
are observed in the 3D structures of PKA, PKB,
CDK and MAP kinase. The Arg in RD doublet
(RD-Arg) is absolutely conserved in all the tyrosine
kinases known to date. The phospho-amino acid
1027
in the activation loop of the tyrosine kinases, however, lacks a direct interaction with the RD-Arg. It
should be noted that among the four residues
equivalent to the contact residues in the Ser/Thr
kinases mentioned above, only two are conserved
as positively charged residues (RD-Arg and the
Arg from the activation segment) in the tyrosine
kinases. The contact residues from the N-terminal
lobe are either apolar or uncharged and lack interactions with the phospho-amino acid (Figures 2(b)
and 4(a) and (b)). The mode of stabilization of
activation segment in the tyrosine kinases to reach
out for the substrate phospho-sites therefore
seems to differ from that of Ser/Thr kinases.
Water mediated interaction of the RD-Arg with
the phospho-amino acid, has been observed in the
crystal structure of LCK.10 A novel cation –p (aromatic ring) interaction11,12 between the RD-R and
the aromatic side-chain of well-conserved Phe
(Phe1186 in IRK) has been identified as a unique
feature (Figure 4(a) and (b)) among the tyrosine
kinases. The critical parameters that define such a
contact include the inter-atomic distance between
the CG, CD, NE, CZ, NH1, or NH2 atoms of arginine and any of the six carbon atoms of the aromatic rings to be less than 4.6 Å.13 Such aromaticcation interactions are characterized by the parallel
alignment of the aromatic ring to the plane of the
guanidium group. The Phe residue located in the
loop joining the activation segment to the aF-helix
further interacts with various hydrophobic residues including Met1176 and Leu1171 (p þ 1 and
p þ 3 substrate-binding pocket) located in the
activation segment of IRK. This Phe is hence likely
to be an important mediator in channeling the conformational changes occurring at the activation
segment to the catalytic loop via the RD-Arg.
Similar interactions are observed between the
equivalent phenylalanine and the hydrophobic
residues in the activation segment of the other
tyrosine kinases (Figure 4(b)). In the receptor
kinase, Ret a M918T mutation leads to multiple
endocrine neoplasia.14 The mutated residue is
equivalent to M1176 of IRK and is involved in the
formation of p þ 3 substrate-binding pocket of
IRK.15 The apolar contacts of M1176 with the Phe
are likely to be directed by the cation – p interactions between the planar ring of Phe and the
RD-Arg. The influence of the aromatic-cation interaction between the RD-Arg and the conserved Phe
residue in activation loop of tyrosine kinases on
substrate binding needs to be further explored.
The extended network of interactions involving
the RD-Arg of the catalytic loop, the highly conserved phenylalanine (F1186) and the hydrophobic
residues (M1176, L1171) can therefore be regarded
as novel “local signal integration motifs”8 unique
to tyrosine kinases. Occurrence of such interactions
in both active and inactive tyrosine kinases as
evident from their crystal structures suggests a
pre-formed substrate-binding site, which is, however, restricted to substrate access by the activation
segment that wraps around the catalytic site in
1028
Review: Phosphorylation Sites in Kinases
Figure 2 (legend opposite)
inactive tyrosine kinases. The cation– p interaction
is also observed between two other highly conserved residues of tyrosine kinases, non-RD Arg
in the catalytic loop (R1136 in IRK) and a Trp residue (W1175 in IRK) in the activation segment. An
analogous interaction between the Lys (K168 in
PKA) and Thr (T201) is found in Ser/Thr kinases
only in their active state (Figure 3). The positively
charged amino acid residues in the catalytic loop
of the tyrosine kinases therefore seek an alternate
mode of stabilization mediated through cation –p
interactions in contrast to the electrostatic and
polar interactions observed for equivalent residues
in the active state of Ser/Thr kinases. The crystal
structures of receptor tyrosine kinases (IRK and
IGFR) in their active state15,16 also reveal contacts
of the phospho-amino acid with two basic residues
from the activation segment. One of these residues
is equivalent to the contact residue (R1155 in IRK)
that forms a part of the phospho-amino acid net-
work conserved in all the protein kinases described
in the previous sections. The other basic residue
(R1164 in IRK) is well conserved in tyrosine kinases
and has very distinct conformations in the active
and inactive structures.
Summary of the differences between Ser/Thr
and Tyr kinases in the permissive phospho-amino
acid network is schematically represented in
Figure 5. From the above discussion it is evident
that Ser/Thr kinase and Tyr kinases exhibit a few
unique features in their phospho-amino acid
network. The amino acid residues (T201 of PKA,
W1175 of IRK) involved in the interactions with
the positively charged residues (K168 of PKA and
R1136 of IRK) in the catalytic loop of Ser/Thr and
Tyr kinases are specifically conserved within the
group of Ser/Thr and Tyr kinases. These distinct
modes of interactions provide a structural basis
for the conservation of residues specific to Ser/
Thr and tyrosine kinase family.17
Review: Phosphorylation Sites in Kinases
1029
Figure 2. Multiple sequence alignment of Ser/Thr and Tyr kinases showing key functional residues. Sequences of (a)
Ser/Thr kinases and (b) tyrosine kinases, with 3D structures and discussed in the text, are shown. Numbers shown in
parentheses in the alignment indicate the number of residues inserted in the corresponding region of the alignment.
Color coding used in the alignment is: conserved residues, shaded gray; catalytic residues, black and bold; contact residues, green; other residues interacting with contact residues and specifically conserved in Ser/Thr or tyrosine kinases,
red.
Figure 3. The phospho-amino
acid network at the activation segment of protein kinase B (1o6l), a
typical RD kinase, is shown. The
phospho-amino acid is colored red,
and the contact residues are shown
in green. Similar color-coding is
used to represent them in subsequent Figures. The conserved
residues, lysine of the catalytic loop
and the threonine (discussed in the
text) that are in contact in the phospho-amino acid in on state are
shown in blue.
1030
Review: Phosphorylation Sites in Kinases
Table 1. List of crystal structures of on and off states of RD kinases requiring phosphorylation for activation, investigated in the current review
Kinase
CAPK
CAPK
CDK
CDK
MAPK
MAPK
Protein kinase B
Protein kinase B
Insulin receptor kinase
Insulin receptor kinase
Insulin-like growth factor 1 receptor kinase
Insulin-like growth factor 1 receptor kinase
Lymphocyte kinase
Src
PDB code
Conformational state
Reference
1atp
1j3h
1hcl
1jst
1erk
2erk
1o6l
1gzn
1ir3
1irk
1k3a
1jqh
3lck
2src
Active
Apoenzyme (inactive)
Inactive
Active
Inactive
Active
Active
Inactive
Active
Inactive
Active
Bis-phosphorylated (partially active)
Active
Inactive
9
27
38
39
40
30
41
42
15
43
16
44
10
22
Further, preferences exhibited by specific contact
residues are observed within Ser/Thr kinases for
their interaction with the phospho-amino acids.
For instance, the interaction of the N-terminal lobe
with the phospho-amino acid is mediated by the
N-terminal contact residues (His87 and His196 in
PKA and protein kinase B (PKB) of AGC subfamily, respectively). The equivalent contact residues in CDK and MAPK of CMGC subfamily,
(namely Thr47 and Arg65) do not interact with the
phospho-amino acid residues. Alternately another
contact residue from the N-terminal lobe of CDK
and MAPK (Arg50 and Arg68) interacts with the
phospho-amino acid (Figure 6). These contact residues provide a link between the N-terminal and
the C-terminal lobes of the kinase.5 Preferences for
such contact residues indicate minor structural
differences conserved within the subfamily.
Various factors including residue environment,
oligomeric state of the catalytically active forms
and intrinsic flexibility of the catalytic core are
likely to contribute to the conformational features
conserved within subgroups of protein kinases.
Non-phospho-amino acid interactions with the
contact residues
The RD-kinases, phosphorylated on the activation segment have well conserved basic residues
(contact residues) forming a part of the phosphoamino acid network. The role of these residues in
the charge neutralization by phospho-amino acid
residues is therefore well established. The available
crystal structures of protein kinases in different
functional states suggest other conserved interactions restricted to specific functional states of distinct families of protein kinases. For instance, the
side-chain of RD-Arg contacts the hydroxyl of tyrosine (Y215 in PKA, Y328 in PKB) located in a loop
that continues as aF-helix (Figure 3), in active
forms of some of the Ser/Thr kinases. The equivalent residue is replaced by hydrophobic residues
incapable of making polar contacts in other RD
types of Ser/Thr kinases like checkpoint kinase
and TGF-b receptor kinase that are regulated by
mechanisms differing from the charge neutralization by phospho-amino acid residues or acidic
residues. In the TGF-bR-1, the phosphorylation at
the conserved glycine-serine (GS) region switches
the binding affinity of the receptor towards the
Smad2 and the binding site is no longer recognized
by the inhibitor protein FKBP12, thus relieving the
inhibition.18 The crystal structures of the above
mentioned enzymes are currently available in the
inhibited or inactive states.19,20 The 3D structures
of these kinases in the on state would help in
elucidating the roles of various conserved residues
in catalysis and regulation. It has to be noted that
the conserved RD-Arg-Tyr pair of Ser/Thr kinases
is formed by residues equivalent to the cation – p
pair forming residues of tyrosine kinases. The
current observations indicate that the location of
residues linking the catalytic loop and the activation loop of Ser/Thr and Tyr kinases are conserved in their primary and tertiary structure
while the nature of interactions between the
residues vary across the two groups of kinases.
The contact residues also contribute to the
stabilization of nucleotide binding residues in the
off state. Such interactions are exemplified by
the salt bridge between Glu of Lys-Glu dyad and
the contact residue from the activation loop of Src
family and Tec families of kinases. Crystal structures of inactive hemapoietic cell kinase,21 Src
kinase22 and BTK23 reveal the interactions of
contact residues (R385 of HCK, Src and R544 of
BTK) with Glu of Lys-Glu dyad. The above interaction (Figure 7) is mediated by the conserved
lysine (K72 in PKA) in the active state. The disruption of the salt-bridge contacts between the Glu of
aC-helix and the contact residue in the activation
segment is likely to mediate further conformational
changes that leads to appropriate disposition of
residues at the catalytic centre upon phosphorylation. Hence, the contact residues also serve as
links between the N-terminal lobe and C-terminal
lobe of protein kinases in Src and BTK family in
their inactive state.
Review: Phosphorylation Sites in Kinases
1031
Figure 4. The phospho-amino
acid in the activation segment of
(a) IRK and (b) LCK and its interactions with the contact residues in
the on state are shown. Aromatic
residues involved in cation – p interactions with RD-Arg and other
basic residues discussed in the text
are shown in blue.
Distinct modes of ATP binding in the on and
off states of Ser/Thr protein kinases
The crystal structures of substrate and ATP
bound ternary complexes of various Ser/Thr
protein kinases including PKA, CDK24,25 and phosphorylase kinase26 have indicated a common binding mode of the substrate peptides in their active
conformations. The backbone of the substrate peptides adopts an extended conformation to align
the hydroxyl group suitably at the catalytic site.
An analysis on the conformation of the co-substrate ATP, bound to active and inactive states of
protein kinases, by pair-wise superposition of ATP
atoms reveals distinct features. Average deviations
of atoms in base, ribose and phosphate groups
1032
Review: Phosphorylation Sites in Kinases
Figure 5. Schematic representation of the interactions of phospho-amino acid in the activation segment of (a) Ser/
Thr and (b) Tyr kinases. PKA (a) and IRK (b) have been chosen as examples to illustrate the phospho-amino acid network in the two groups of protein kinases. Distinct nature of interactions with RD-Arg, characterized by electrostatic
interactions in Ser/Thr kinases and cation– p (aromatic) interactions in tyrosine kinases, stabilize the activation segment. Unlike in Ser/Thr kinases, N-terminal helix (aC-helix) of tyrosine kinases lacks interactions with the phosphoamino acid. The arrows (blue) indicate the residues involved in cation – p (aromatic) interactions in tyrosine kinases.
arrived at from all pairwise superpositions of ATP
molecules in kinases are represented in Figure 8.
ATP displays similar conformation in the on
state of Ser/Thr protein kinases as indicated by
the lower deviation of their adenine ring, ribose
and phosphate groups compared to ATP molecules
in the inactive states of kinases (Figure 8). This
feature is consistent with appropriate disposition
Figure 6. Phospho threonine
(pT 183) at the activation segment
of active MAPK and its interactions
with specific contact residues are
shown. Arg65 (blue) equivalent to
His196 of PKB (Figure 3) is drifted
away from the catalytic site and
lacks interactions with pT183.
Review: Phosphorylation Sites in Kinases
1033
Figure 7. 3D structure of Src
kinase (2Src) in an inhibited state.
The interaction between residues
involved in Lys-Glu dyad (red) in
the active state and residues from
the activation segment (blue) is
shown.
Figure 8. Extent of deviation of conformation of ATP bound to active and inactive states of protein kinases. The average deviation (Å) of equivalent atoms of ATP observed for adenine, ribose and phosphate groups among active forms
(red) and between active and inactive forms (dark green) is shown.
1034
of the gamma phosphate groups for inline phospho-transfer.
ATP molecules bound to inactive forms of protein kinases show wide variations in the conformation of their chemical groups. Although the
adenine ring and the ribose groups of ATP deviate
least across active and inactive states, deviations
greater than 1 Å are observed in the equivalent
atoms of the phosphate groups of the ATP. The
beta and gamma phosphate groups are highly
mobile with equivalent atoms deviating by greater
than 3 Å.
These variations in the conformation of ATP are
likely to arise form the large conformational
changes accompanying the key residues involved
in ATP binding and coordination with the metal
ions (Mg2þ, Mn2þ) during transition into different
states. ATP binding residues of the kinase are
localized in distinct regions of the fold that
includes the Gly-loop, hinge region, catalytic loop
and the activation segment. In CDK the key residues comprising the ATP-binding pocket include
Gly13, Thr14, Val18 of Gly loop; Lys33 of Lys-Glu
dyad; Phe80, Glu81, Phe82 and Leu83 of the hinge
region; Asn132 of the catalytic loop and Asp145 of
the activation segment. These residues vary in
their extent of interactions with ATP in their active
and inactive states. While some of these residues
(G13, T14, F80, D145) are involved in ATP binding
only in the active state, other residues (F82, L83)
interact with ATP in the inactive state. The remaining residues participate in ATP binding in both
states of activity.
High mobility of phosphate groups enables their
co-ordination with the metal ions that are displaced due to the variation in the positions of
residue ligands (N132, D145) located in the catalytic and activation loop between the two conformational states of kinases.
The atoms of ATP bound to active form of
kinases in general can be well superimposed.
Large variation is, however, observed in the conformation of ribose and phosphate groups of ATP
bound to phospho-CDK/cyclin complex in comparison with ATP bound to active states of other
Ser/Thr kinases. Accommodation of ATP in altered
conformations within the structurally similar
catalytically active scaffold without hindering
phospho-transfer therefore seems to be influenced
by the binding of cyclin to CDK.
The conformations of ATP bound to Ser/Thr
kinases and tyrosine kinases in the on state also
varies to a large extent. The RTKs lack the formation of a correct Lys-Glu ion-pair in their active
states as described in a later section. The nature
of stabilization of the activation segment of tyrosine kinases in the on state is also distinct from
that of Ser/Thr kinases as described earlier. High
sequence divergence is observed in the nucleotide
binding pockets of Ser/Thr and Tyr kinases. These
distinct functional and structural features of Tyr
kinases are hence likely to influence the mode of
ATP binding in the tyrosine kinases. The different
Review: Phosphorylation Sites in Kinases
features appear to play an important role in the
distinct nature of co-ordination of phosphate
groups of ATP with the conserved lysine and
metal ions.
Influence of phosphorylation and ATP binding
on closed state of protein kinases
Appropriate spatial orientation of the ATP
binding residues and the relative disposition of
catalytic residues in the activation and catalytic
loop are characteristic to the closed state of
protein kinases. The aC-helix is a key structural
element in the N-terminal lobe mediating the
conformational changes occurring at the catalytic
center through an ion pair (Lys72-Glu91 in PKA)
that bridges phosphate groups of ATP. The charge
neutralization of the basic cluster of residues in the
active site5 is another critical feature in RD kinases
regulated by phosphorylation as described earlier.
The crystal structure of the apo-enzyme of phosphorylated PKA27 reveals the electrostatic interactions of the phospho-amino acid similar to its
ternary complex.
However, the Lys and Glu residues are spatially
distant, incapable of salt bridge formation and
coordination with a- and b-phosphate groups of
ATP. Phosphorylation of the activation segment in
PKA therefore appears to alter the active site
locally, with least influence on the formation of
ion pair in the N-terminal lobe.
In contrast to the Ser/Thr kinases, the receptor
tryosine kinases lack the Lys-Glu ion-pair interaction in their active states as observed in the 3D
structures of insulin receptor15 and IGF receptor
kinases.16 However, the phospho-amino acid in
the activation segment is capable of interacting
with the basic residues in its vicinity in a unique
way as described in the previous sections.
In the conformationally restrained state of
c-Abl28,29 the Lys-Glu ion pair interaction is
retained. However, the residues at the catalytic
site are not aligned for phospho transfer, as the
contact residues are not drawn towards Tyr412,
which is phosphorylated for complete activation
of the kinase.
The formation of Lys-Glu ion pair and conformational changes at the active site accompanying
the activation process are hence suggested to vary
across different protein kinases. The mechanisms
of acquisition of a closed, functionally competent
state in protein kinases is therefore suggested to
vary based on the extent of reorganization of the
active site necessary to initiate phospho transfer.
Interactions of “secondary” phospho-amino
acid residues in protein kinases
The phosphorylation occurs at multiple sites on
protein kinases providing additional controls on
their activity. In MAP kinases of CMGC subfamily,
the successive dual phosphorylation of the Thr
and Tyr residues in TXY motif of the activation
Review: Phosphorylation Sites in Kinases
segment results in local and global conformational
changes30 realigning the positively charged contact
residues close to the phospho threonine (pT183).
The second dianionic site unique to MAPK (Figure
9(a)) created by the phosphorylation of Tyr residue
also interacts with two basic residues (Arg189,
Arg192). Glycogen synthase kinase-3 (GSK-3),
another member of CMGC subfamily is usually
kept active in cells and is known to phosphorylate
a wide range of cellular proteins.31 PKB mediated
phosphorylation on the N-terminal serine residue
of GSK-3b leads to its inactivation. GSK-3b is
unique in its choice of substrates with higher
preferences for phosphorylated (primed) substrates. The phosphorylation on the activation
segment for regulation of this enzyme is nonobligatory. Analogous interactions are observed in
the 3D structure32 of GSK between the phospho-
1035
tyrosine (pY) 416 (only phospho-amino acid) in
the activation segment and Arg220 and Arg223
(Figure 9(b)). The basic residues hence are likely
to play a major role in accommodating the phospho-amino acid on the primed substrates similar
to their contacts with pY416 in the absence of the
substrate.
The four main groups of CMGC kinases (CDK,
MAPK, glycogen synthase kinase (GSK) and casein
kinase-2 (CK2)) show high conservation of basic
residues in equivalent positions with the exception
of CDK. An apolar residue (Leu166) replaces the
residue equivalent to the Arg189 (MAPK) in CDK
while the other basic residue is well conserved.
However, the role of these residues in CDK that
possess mechanisms of activation distinct from
MAPK and GSK is unclear. CK2 is a constitutively
active kinase and no phosphorylation at the
Figure 9. The interactions of secondary phospho-amino acid residues in the active and inactive states of protein
kinases. (a) Active state of MAPK, (b) phospho-glycogen synthase kinase (GSK), (c) inactive state of c-Src kinase
(inactive) and (d) inactive state of hemapoietic cell kinase (HCK).
1036
catalytic subunit is so far documented for its
regulation. It has to be noted that the Sky1p33 of
Saccharomyces cerevisiae (a non-RD kinase) is the
only other protein kinase in addition to the RD
kinases of the CMGC group, with basic residues
conserved at equivalent positions.
The inhibitory phosphorylation site of Src family
protein kinases is located C-terminal to the kinase
domain. Upon phosphorylation the phospho-tyrosine (Tyr527) docks into an intramolecular site
located in the SH2 domain.22,34 This interaction is
mediated by an ion pair between the wellconserved pair of Arg residues (R155, R175) in the
SH2 domain and the phosphate (Figure 9(c)). The
crystal structure of inactive HCK,21 another
member of Src family of kinases reveals analogous
interactions with a pair of basic residues in its
SH2 domain. The basic residues include an Arg
equivalent to the R175 of Src while the other basic
residue (K203) is located in a different strand
unlike R155 in the Src (Figure 9(d)). Thus, different
basic residues interact with the inhibitory pTyr in
HCK and Src as summarized in a schematic Figure
(Figure 10). These observations suggest that the
spatial location of the phospho-amino acid interacting residues is conserved, while their positions
in the primary structure vary.
The C-terminal Src kinase (CSK) is similar to Src
kinases in their modular organization with SH3,
SH2 and kinase domains. The in-built mechanism
of regulation is quite distinct from that of Src
kinases lacking phospho sites in the activation segment and the C-terminal tail. While basic residues
interacting with inhibitory phospho-sites in Src
are well conserved in CSK, a salt bridge exists
between one of the basic residues and Asp residue
located in the helix of SH2 domain in their inactive
state35 analogous to the contact of phospho-amino
acid. These observations indicate that the phosphoamino acid network at the inhibitory phospho
sites of Src show preferences for the basic residues
as exemplified by HCK and Src kinases. Among
the cytoplasmic tyrosine kinases, with similar
domain organization cAbl and CSK lack inhibitory
phospho sites (cAbl and CSK). Conserved nega-
Review: Phosphorylation Sites in Kinases
tively charged residues of cAbl and CSK (E123
in cAbl) form ion pairs with the basic residues
equivalent to the inhibitory phospho site interacting residues in the off state.
Coupling of inhibitory phospho-sites of CDK
with the Lys-Glu dyad
Phosphorylation on conserved Thr and Tyr
(Thr14 and Tyr15) residues in the Gly rich loop of
CDK by wee1 kinase36 leads to its inactivation.
The crystal structures of CDKs available in inhibited and inactive states reveal these phosphorylation sites to be distant from the aC-helix.
However, the partially active cyclin bound forms
of CDK and the fully active phospho-CDK/cyclin
complex show contacts between the hydroxyl
group of Tyr15 and the Glu51 of the Lys-Glu dyad
that anchors ATP. The proximity of the Tyr15 (in
glycine-loop) and Glu51 (Figure 11) induced by
cyclin binding, can cause a repulsion between like
charges upon phosphorylation of Tyr15 drifting
the aC-helix away from Gly-rich loop. The electrostatic repulsion is therefore likely to mediate the
transition of the partially active and fully active
CDKs to the inactive states.
Crystal structure of CDK phosphorylated at the
inhibitory sites should therefore clarify the role of
interactions of the Lys-Glu dyad with inhibitory
phospho sites in the Gly-rich loop.
Conclusions
The active state of Ser/Thr and Tyr kinases
adopt grossly similar structures that enable efficient catalysis on diverse substrates. Phosphorylation at the activation segment is the major means
of regulation of most of the RD class of kinases.
Interactions subsequent to phosphorylation at the
active site of these kinases facilitate substrate
access and phospho-transfer. While the chemical
environment of the active sites in the vicinity of
phospho-amino acid is well conserved, the spatial
location, extent and mode of interactions vary
Figure 10. Schematic representation of interactions of inhibitory
phospho-tyrosine in the Src family
of protein kinases. (left) c-Src and
(right) hemapoietic cell kinase
(HCK). The network of phosphoamino acid is comprised of distinct
positively charged residues well
conserved across the two protein
kinases.
Review: Phosphorylation Sites in Kinases
1037
Figure 11. 3D structure of cyclin
dependent kinase showing interactions between Tyr15 (red), the
inhibitory phosphorylation site
and conserved Glu51 (red) of Lys
(green)-Glu dyad in cyclin boundphosphorylated complex (1JST).
across Ser/Thr and Tyr kinases. The Arg of the
RD motif stabilizes the activation segment by
electrostatic interaction with the phospho-amino
acid in most Ser/Thr kinases in their on state. In
contrast in the Tyr kinase the RD-Arg lacks the
conserved interaction with the phospho-amino
acid and alternately makes cation – p interactions
in on and off states with phenylalanine well conserved in known Tyr kinases. The phenylalanine
further extends its interaction to the substratebinding pocket and is hence likely to be important
for the formation of substrate binding sites.
Crystal structures of Ser/Thr kinases in the
active state also reveal conservation of polar interactions of RD-R with conserved tyrosine that is
uniquely conserved among RD kinases regulated
by negative charge or phospho-amino acid in the
activation segments. While the role of such interactions is currently unclear, it is likely to be another
feature of the active site of protein kinases specifically conserved in the on state.
The significance of the stabilizing interactions of
the contact residues with the phospho-amino acid
in the active state of kinases is well established.
Ion pair between the contact residue and the LysGlu dyad in the off-state of Src kinases and BTK
suggest coupling of the activation segment and
aC-helix, which communicates the changes to the
catalytic center subsequent to phosphorylation.
Preferences for interactions of the phosphoamino acid with specific contact residues in
aC-helix, are observed within the group of Ser/
Thr kinases as exemplified by AGC and CMGC
family of kinases. The variation in orientation of
aC-helix in distinct functional states of these subgroups of kinases and the intrinsic flexibility
associated with the catalytic core for such movements, appear to be critical for the choice of
residues in the phospho-amino acid network.
Inhibitory phosphorylation sites of Src family of
kinases also create a cluster of positively charged
residues, which are usually exposed in the on
states. Distinct set of basic residues, however,
participate in such interactions in the c-Src and
HCK family of kinases.
The tyrosine in the Gly-rich loop of CDKs is
proximal to Glu of Lys-Glu dyad in the active and
partially active structures. Phosphorylation of the
Tyr in the cyclin bound forms of CDKs leading to
inhibition of CDK is hence suggested to result
from the repulsion between Glu and the dianionic
charge.
The available 3D structures of protein kinases
have enabled the identification of a subset of
unique features associated with the active state
of Ser/Thr and tyrosine kinases despite many
common structural features and mechanisms of
phospho transfer. The participation of residues
conserved specifically in the two groups of kinases
in unique interactions provides a structural basis
for their conservation. While interactions of the
phospho-amino acid residues located at various
sites in the 3D-scaffold of the protein kinases with
positively charged residues is observed, the extent
of neutralization by the dianionic charge and the
spatial location of the positively charged residues
may vary.
ATP bound to Ser/Thr kinases in the on state,
1038
in general, adopt similar conformation of adenine,
ribose and phosphate groups. The conformation of
ATP varies between active and inactive forms of
ATP, with the phosphate groups showing the large
deviation in their relative orientation. Variations
are, however, observed in active states of other
protein kinases (CDK) which might be partially
influenced by additional factors like the interactions of the kinases with regulatory proteins.
The vast number of crystal structures of RDkinases in distinct conformational states have
aided in the identification of conserved and varied
functional features at their active sites subsequent
to phosphorylation. The number of 3D structures
currently available for protein kinases regulated
by other modes of regulation in on and off states
is limited. The emergence of crystal structures of
non-RD kinases in their on state will enhance the
current understanding of the factors stabilizing
their active sites.
Review: Phosphorylation Sites in Kinases
11.
12.
13.
14.
15.
Acknowledgements
This research is supported by the Wellcome
Trust, London, in the form of Senior Fellowship in
Biomedical Sciences to N.S. A.K. is supported by
Council of Scientific and Industrial Research,
New Delhi. G.P. is a visiting student trainee to the
Indian Institute of Science.
16.
17.
18.
References
1. Schenk, P. W. & Snaar-Jagalska, B. E. (1999). Signal
perception and transduction: the role of protein
kinases. Biochim. Biophys. Acta, 1449, 1 – 24.
2. Cohen, P. (2000). The regulation of protein function
by multisite phosphorylation—a 25-year update.
Trends Biochem. Sci. 25, 596– 601.
3. Hunter, T. (2000). Signaling—2000 and beyond. Cell,
100, 113 – 127.
4. Huse, M. & Kuriyan, J. (2002). The conformational
plasticity of protein kinases. Cell, 109, 275–282.
5. Johnson, L. N., Noble, M. E. & Owen, D. J. (1996).
Active and inactive protein kinases: structural basis
for regulation. Cell, 85, 149– 158.
6. Kobe, B. & Kemp, B. E. (1999). Active site-directed
protein regulation. Nature, 402, 373– 376.
7. Johnson, L. N., Lowe, E. D., Noble, M. E. & Owen,
D. J. (1998). The eleventh datta lecture. The structural
basis for substrate recognition and control by protein
kinases. FEBS Letters, 430, 1 – 11.
8. Johnson, L. N. & Lewis, R. J. (2001). Structural basis
for control by phosphorylation. Chem. Rev. 101,
2209– 2242.
9. Knighton, D. R., Zheng, J. H., Ten Eyck, L. F., Ashford, V. A., Xuong, N. H., Taylor, S. S. & Sowadski,
J. M. (1991). Crystal structure of the catalytic subunit
of cyclic adenosine monophosphate-dependent
protein kinase. Science, 253, 407– 414.
10. Yamaguchi, H. & Hendrickson, W. A. (1996). Structural basis for activation of human lymphocyte
19.
20.
21.
22.
23.
24.
25.
kinase LCK upon tyrosine phosphorylation. Nature,
384, 484– 489.
Singh, J. & Thornton, J. M. (1990). SIRIUS. An automated method for the analysis of the preferred packing arrangements between protein groups. J. Mol.
Biol. 211, 595– 615.
Flocco, M. M. & Mowbray, S. L. (1994). Planar stacking interactions of arginine and aromatic side-chains
in proteins. J. Mol. Biol. 235, 709– 717.
Momany, F. A., Carruthers, L. M., McGuire, R. F. &
Scheraga, H. A. (1974). Intermolecular potentials
and applications to the packing configurations and
lattice energies in crystals of hydrocarbons, carboxylic acids, amines and amides. J. Phys. Chem. 78,
1595– 1620.
Hofstra, R. M., Landsvater, R. M., Ceccherini, I.,
Stulp, R. P., Stelwagen, T., Luo, Y., Pasini, B.,
Hoppener, J. W., van Amstel, H. K. & Romeo, G.
(1994). A mutation in the RET protooncogene associated with multiple endocrine neoplasia type 2B
and sporadic medullary thyroid carcinoma. Nature,
367, 375– 376.
Hubbard, S. R. (1997). Crystal structure of the activated insulin receptor tyrosine kinase in complex
with peptide substrate and ATP analog. EMBO J. 16,
5572– 5581.
Favelyukis, S., Till, J. H., Hubbard, S. R. & Miller,
W. T. (2001). Structure and autoregulation of the
insulin-like growth factor 1 receptor kinase. Nature
Struct. Biol. 8, 1058– 1063.
Singh, J. (1994). Comparison of conservation within
and between the Ser/Thr and Tyr protein kinase
family: proposed model for the catalytic domain of
the epidermal growth factor receptor. Protein Eng. 7,
849– 858.
Huse, M., Muir, T. W., Xu, L., Chen, Y. G., Kuriyan, J.
& Massague, J. (2001). The TGF beta receptor activation process: an inhibitor- to substrate-binding
switch. Mol. Cell, 8, 671– 682.
Huse, M., Chen, Y. G., Massague, J. & Kuriyan, J.
(1999). Crystal structure of the cytoplasmic domain
of the type I TGF beta receptor in complex with
FKBP12. Cell, 96, 425–436.
Chen, P., Luo, C., Deng, Y., Ryan, K., Register, J.,
Margosiak, S., Tempczyk-Russell, A., Nguyen, B.,
Myers, P., Lundgren, K., Kan, C. C. & O’Connor,
P. M. (2000). The 1.7 Å crystal structure of human
cell cycle checkpoint kinase Chk1: implications for
Chk1 regulation. Cell, 100, 681– 692.
Sicheri, F., Moarefi, I. & Kuriyan, J. (1997). Crystal
structure of the Src family tyrosine kinase Hck.
Nature, 385, 602 –609.
Xu, W., Doshi, A., Lei, M., Eck, M. J. & Harrison, S. C.
(1999). Crystal structures of c-Src reveal features of
its autoinhibitory mechanism. Mol. Cell, 3, 629– 638.
Mao, C., Zhou, M. & Uckun, F. M. (2001). Crystal
structure of Bruton’s tyrosine kinase domain
suggests a novel pathway for activation and provides insights into the molecular basis of X-linked
agammaglobulinemia.
J.
Biol.
Chem.
276,
41435– 41443.
Brown, N. R., Noble, M. E., Endicott, J. A. & Johnson,
L. N. (1999). The structural basis for specificity of
substrate and recruitment peptides for cyclindependent kinases. Nature Cell Biol. 1, 438– 443.
Brown, N. R., Noble, M. E., Lawrie, A. M., Morris,
M. C., Tunnah, P., Divita, G., Johnson, L. N. &
Endicott, J. A. (1999). Effects of phosphorylation of
Review: Phosphorylation Sites in Kinases
26.
27.
28.
29.
30.
31.
32.
33.
34.
threonine 160 on cyclin-dependent kinase 2 structure
and activity. J. Biol. Chem. 274, 8746– 8756.
Lowe, E. D., Noble, M. E., Skamnaki, V. T.,
Oikonomakos, N. G., Owen, D. J. & Johnson, L. N.
(1997). The crystal structure of a phosphorylase
kinase peptide substrate complex: kinase substrate
recognition. EMBO J. 16, 6646 –6658.
Akamine, P., Madhusudan, Wu, J., Xuong, N. H., Ten
Eyck, L. F. & Taylor, S. S. (2003). Dynamic features of
cAMP-dependent protein kinase revealed by apoenzyme crystal structure. J. Mol. Biol. 327, 159– 171.
Hantschel, O., Nagar, B., Guettler, S., Kretzschmar, J.,
Dorey, K., Kuriyan, J. & Superti-Furga, G. (2003). A
myristoyl/phosphotyrosine switch regulates c-Abl.
Cell, 112, 845– 857.
Nagar, B., Hantschel, O., Young, M. A., Scheffzek, K.,
Veach, D., Bornmann, W., Clarkson, B., SupertiFurga, G. & Kuriyan, J. (2003). Structural basis for
the autoinhibition of c-Abl tyrosine kinase. Cell, 112,
859–871.
Canagarajah, B. J., Khokhlatchev, A., Cobb, M. H. &
Goldsmith, E. J. (1997). Activation mechanism of the
MAP kinase ERK2 by dual phosphorylation. Cell,
90, 859– 869.
Welsh, G. I., Miyamoto, S., Price, N. T., Safer, B. &
Proud, C. G. (1996). T-cell activation leads to rapid
stimulation of translation initiation factor eIF2B and
inactivation of glycogen synthase kinase-3. J. Biol.
Chem. 271, 11410 –11413.
Bax, B., Carter, P. S., Lewis, C., Guy, A. R., Bridges,
A., Tanner, R., Pettman, G., Mannix, C., Culbert,
A. A., Brown, M. J., Smith, D. G. & Reith, A. D.
(2001). The structure of phosphorylated GSK-3beta
complexed with a peptide, FRATtide, that inhibits
beta-catenin phosphorylation. Structure (Camb), 9,
1143– 1152.
Nolen, B., Ngo, J., Chakrabarti, S., Vu, D., Adams,
J. A. & Ghosh, G. (2003). Nucleotide-induced conformational changes in the Saccharomyces cerevisiae SR
protein kinase, Sky1p, revealed by X-ray crystallography. Biochemistry, 42, 9575– 9585.
Young, M. A., Gonfloni, S., Superti-Furga, G., Roux,
B. & Kuriyan, J. (2001). Dynamic coupling between
the SH2 and SH3 domains of c-Src and Hck
underlies their inactivation by C-terminal tyrosine
phosphorylation. Cell, 105, 115 – 126.
1039
35. Ogawa, A., Takayama, Y., Sakai, H., Chong, K. T.,
Takeuchi, S., Nakagawa, A., Nada, S., Okada, M. &
Tsukihara, T. (2002). Structure of the carboxylterminal Src kinase, Csk. J. Biol. Chem. 277,
14351 – 14354.
36. Russell, P. & Nurse, P. (1987). Negative regulation of
mitosis by wee1 þ , a gene encoding a protein kinase
homolog. Cell, 49, 559– 567.
37. Evans, S. V. (1993). SETOR: hardware-lighted threedimensional solid model representations of macromolecules. J. Mol. Graph. 11, 134– 138. see also pp.
127 –128.
38. Schulze-Gahmen, U., Brandsen, J., Jones, H. D.,
Morgan, D. O., Meijer, L., Vesely, J. & Kim, S. H.
(1995). Multiple modes of ligand recognition: crystal
structures of cyclin-dependent protein kinase 2 in
complex with ATP and two inhibitors, olomoucine
and isopentenyladenine. Proteins: Struct. Funct.
Genet. 22, 378– 391.
39. Russo, A. A., Jeffrey, P. D. & Pavletich, N. P. (1996).
Structural basis of cyclindependent kinase activation
by phosphorylation. Nature Struct. Biol. 3, 696– 700.
40. Zhang, F., Strand, A., Robbins, D., Cobb, M. H. &
Goldsmith, E. J. (1994). Atomic structure of the MAP
kinase ERK2 at 2.3 Å resolution. Nature, 367,
704 –711.
41. Yang, J., Cron, P., Good, V. M., Thompson, V.,
Hemmings, B. A. & Barford, D. (2002). Crystal structure of an activated Akt/protein kinase B ternary
complex with GSK3-peptide and AMP-PNP. Nature
Struct. Biol. 9, 940– 944.
42. Yang, J., Cron, P., Thompson, V., Good, V. M., Hess,
D., Hemmings, B. A. & Barford, D. (2002). Molecular
mechanism for the regulation of protein kinase B/
Akt by hydrophobic motif phosphorylation. Mol.
Cell, 9, 1227– 1240.
43. Hubbard, S. R., Wei, L., Ellis, L. & Hendrickson, W. A.
(1994). Crystal structure of the tyrosine kinase
domain of the human insulin receptor. Nature, 372,
746 –754.
44. Pautsch, A., Zoephel, A., Ahorn, H., Spevak, W.,
Hauptmann, R. & Nar, H. (2001). Crystal structure
of bisphosphorylated IGF-1 receptor kinase: insight
into domain movements upon kinase activation.
Structure (Camb), 9, 955– 965.
Edited by J. Thornton
(Received 11 February 2004; received in revised form 21 April 2004; accepted 22 April 2004)