c-Src Tyrosine Kinases
Marissa Bradbury
Shaileja Chopra
Shelley Crispin
Srilalitha Kuruganti
Introduction:
Heterotrimeric G proteins transduce signals from cell surface receptors to modulate
the activity of cellular effectors. Src- the product of the first characterized protooncogene and the first identified protein tyrosine kinase, plays a critical role in the signal
transduction of G-protein coupled receptors. The heterotrimeric G protein consists of
three subunits: ,  and . Based on the differences in their genes, 20 , 6  and 12 
subunits have been identified. Their molecular weights are in the following ranges:

 subunit: 39 - 46 kD

subunit: 35 - 39 kD

subunit: ~ 8 kD
The subunit: 35 - 39 kD and subunit: ~ 8 kDs are tightly associated and can be
regarded as one functional unit. G proteins function as molecular binary switches with
their biological activity determined by the bound nucleotide.
Tyrosine phosphorylation has been implicated in the regulation of a variety of
biological responses including cell proliferation, migration, differentiation, and survival.
The protein tyrosine kinases involved in mediating these responses, as well as the
receptors that activate them, encompass a diverse spectrum of proteins. One family of
cytoplasmic tyrosine kinases capable of communicating with a large number of different
receptors is the Src protein tyrosine kinase family (Src PTKs).
Structural domains of src-kinases:
Src PTKs are 52–62 kDa proteins composed of six distinct functional regions .
(a) the Src homology (SH) 4 domain,
(b) the unique region,
(c) the SH3 domain,
(d) the SH2 domain,
(e) the catalytic domain, and
(f) a short negative regulatory tail
The SH4 domain is a 15-amino acid sequence that contains signals for lipid
modification of Src PTKs. The glycine at position 2 is important for addition of a myristic
acid moiety, which is involved in targeting Src PTKs to cellular membranes. The three
domains that follow the unique region represent modular structures found in many classes
of cellular proteins. The SH3 and SH2 domains are protein-binding domains present in
lipid kinases, protein and lipid phosphatases, cytoskeletal proteins, adaptor molecules,
transcription factors, and other proteins. The catalytic domain possesses tyrosine-specific
protein kinase activity.
The SH3 domains of Src PTKs are composed of 50 amino acids. A second modular
domain that also controls the repertoire of proteins interacting with Src PTKs is the SH2
domain. Binding interactions mediated by the SH2 domain function in regulating the
catalytic activity of Src PTKs, as well as the localization of Src or its binding proteins. All
SH2 domains bind to short contiguous amino acid sequences containing phosphotyrosine,
and the specificity of individual SH2 domains lies in the 3–5 residues following the
phosphotyrosine (+1, +2, +3, etc). Amino acids preceding phosphotyrosine may also be
important for regulating binding affinity. Structural studies on Src family SH2 domains
have shown that the ligand-binding surface of SH2 domains is composed of two pockets.
One pocket contacts the phosphotyrosine; the other pocket contacts the +3 amino acid
residue following the phosphotyrosine. Src family kinases show a preference for leucine
at this position. Examples of proteins shown to interact with the Src SH2 domain in vivo
include the focal adhesion protein FAK (focal adhesion kinase), p130cas, p85 PI 3-K, and
p68sam.
Expression of Src Family Kinases:
The Src PTKs can be subdivided into three groups based on their general pattern
of expression. Src, Fyn, and Yes are expressed in most tissues; however, individual
kinases are expressed at elevated levels in certain cell types and some of these genes are
expressed as alternatively spliced mRNAs in specific cell types. For example, Src is
expressed ubiquitously; however, platelets, neurons, and osteoclasts express 5–200-fold
higher levels of this protein
Table 1. Expression of Src family kinases
Src
Ubiquitous; two neuron-specific isoforms
Fyn
Ubiquitous; T cell-specific isoform (Fyn T)
Yes
Ubiquitous
Yrka
Ubiquitous
Lyn
Brain, B-cells, myeloid cells; two alternatively spliced forms
Hck
Myeloid cells (two different translational starts)
Fgr
Myeloid cells, B-cells
Blk
B-cells
Lck
T-cells, NK cells, brain
Frk subfamily Primarily epithelial cells
Frk/Rak
Iyk/Bsk
a
Only found in chickens.
Activation of Src Kinases:
The SH2 and SH3 domains play a central role in regulating Src PTK catalytic
activity. High-resolution crystal structures of human Src and Hck, in their repressed state,
have provided a structural explanation for how intramolecular interactions of the SH3 and
SH2 domains stabilize the inactive conformation of these kinases. The crystal structures
include the SH3, SH2, and catalytic domains, and the negative regulatory tail. Both the
SH3 and SH2 domains lie on the side of the kinase domain opposite the catalytic cleft.
The SH3 and SH2 domains repress the kinase activity by interacting with amino acids
within the catalytic domain, as well as with residues N-terminal and C-terminal,
respectively, to the catalytic domain.
Figure. Mechanisms involved in activation of Src family kinases. The left panel shows a
model of the structure of inactivated Src PTKs that are phosphorylated on the C-terminal
tyrosine (Y527 in this model of Src). This model is based on the crystal structures of Src
and Hck. The middle panel shows possible mechanisms involved in activation of Src
PTKs. Y416 represents the autophosphorylation site in the activation loop of Src. The
right panel represents a model for the activated state of Src in which the intramolecular
interactions of the SH3 and SH2 domains are disrupted.
The SH3 domain interacts with sequences in the catalytic domain, as well as with
sequences in the linker region that lies between the SH2 and catalytic domains. Although
the linker region contains only a single proline residue, these sequences form a lefthanded PPII helix and bind the SH3 domain in the same orientation as class II ligands.
Two regions of the SH3 domain that flank the hydrophobic binding surface make contacts
with the catalytic domain. Thus interactions with the linker region and the kinase domain
are likely to account for the SH3 domain's role in negatively regulating the catalytic
activity of Src PTKs.
The SH2 domain interacts with pTyr 527 (Src) and adjacent residues in the negative
regulatory tail. Y527 in c-Src, and the corresponding tyrosine in other Src PTKs, are the
primary sites of tyrosine phosphorylation in vivo. This residue is phosphorylated by the
cytoplasmic tyrosine kinase Csk. Several lines of evidence indicate that loss of Y527
phosphorylation leads to activation of Src catalytic activity:
(a) Mutation of Y527 results in constitutive activation of c-Src,
(b) Y527 and several amino acids surrounding this residue are deleted in v-Src and
similar truncations of c-Src cause activation of this enzyme.
(c) Disruption of the csk gene results in activation of at least three Src PTKs
These results and others support a model whereby Csk-mediated tyrosine
phosphorylation of the C-terminal tail promotes an intramolecular interaction between the
SH2 domain and the phosphorylated tail, keeping the kinase in a closed, inactive
conformation. These include displacement of the intramolecular interactions of the SH2
or SH3 domains by high-affinity ligands or modification of certain residues,
dephosphorylation of pY527 by a tyrosine phosphatase, or phosphorylation of Y416. In
summary, the modular domains of Src PTKs endow these kinases with the ability to be
regulated by and to communicate with a diverse group of proteins.
Researchers have spent decades trying to elucidate the structure of Src tyrosine
kinases. Finally, in 1997 two laboratories published the crystal structure of c-Src tyrosine
kinase in its inactive conformation. Both structures agreed with each other and a model
has been proposed for the active conformation of c-Src yet no crystal structure exists of
the kinase in this conformation. Below is a diagram depicting the organization of the cSrc tyrosine kinase domains (Engen J., 2002).
The SH2 and SH3 domains are connected via a linker and it is through these domains that
protein-protein interactions are mediated during signal transduction. The N terminal
sequence is responsible for anchoring the protein to the cell membrane while the function
of the unique domain remains unknown. The SH3 and SH2 domains are responsible for
binding proline-rich ligands and phosphorylated tyrosine sequences respectively. The
linker domain binds intramolecularly with SH3 and associates with the catalytic domain.
The catalytic domain itself is comprised of two lobes, the N lobe and the C lobe. The
activation loop is responsible for regulating enzymatic activity while the C-terminal tail,
when phosphorylated, can bind to the SH2 domain. Below is the overall structure of the
inactive form of c-Src tyrosine obtained from crystal structure analysis.
In the active state, the SH2 domain interacts with phosphorylated Tyr 527 on the
face of the catalytic domain. There is an intramolecular interaction in which the linker
connecting the SH2 domain to the catalytic domain forms a type II polyproline helix to
which the SH3 domain is bound. This resembles closely the standard binding mode of
SH3 domains. This interaction of the phosphorylated Tyr 527 tail and the SH2 domain
impedes the ability of the protein to phosphorylate Tyr 416 in the activation loop. The
SH2 and SH3 domains do not directly block the active site of the catalytic domain.
Instead, the catalytic activity is lost due to conformational changes at the active site of the
catalytic domain. An alpha helix, C, borders the active site and is rotated outward in the
inactive form resulting in the movement of the glutamate side chain out of the active site.
Thus, in the inactive state, the activation loop is restructured such that the substratebinding site is blocked. This change in structure also results in displacement of Asp 404,
so that it can no longer properly coordinate a critical Mg++ ion. Below is a depiction of
the inactive versus active conformation of c-Src tyrosine kinase (Young M. et al., 2001).
Below is a ribbon diagram depicting the difference between the catalytic domain in the
active and inactive conformation (Engen J., 2002).
Experimental evidence:
`Targeted’ molecular dynamics trajectories were generated, in which the
conformation of the activation loop is driven toward the active form artificially, starting
from the inactive form. These simulations have shown that the SH2 and SH3 domains are
coupled dynamically when the SH2 domain is bound to the phosphorylated Tyr 527. The
simulations also indicate that a short connector region between the SH2 and SH3 domains
has direct implications on the regulatory mechanism. Structural studies indicate that the
structure of the connector is stabilized by the formation of hydrogen bonds. The
connector contains eight residues that adopt a  turn followed by a single turn of the 310
helix, lined by hydrogen bonds.
The en-bloc movements of the SH2 and the SH3 domains were quantitated by
calculating the cross-correlation coefficients for the fluctuations in the positions of the
C atoms during the simulation. The results showed that the SH2 and the SH3 domains
were highly correlated to the C-terminal tail and the SH2 kinase linker respectively. The
SH3 domain was found to be strongly correlated to the connector, which is turn is
correlated to the SH2 domain. It was also demonstrated that when the phosphorylated tail
is connected to the SH2 domain (inactive state), the SH3 domain is seen to fluctuate very
little in position relative to the N-terminal lobe of the kinase. Two different simulations
generated from the inactive and the assembled state (with the C-terminal tail in
dephosphorylated form) indicate that in both cases the fluctuations within the SH2 and
SH3 domains become less correlated. In addition, it was found that increased motions in
the SH2 domain leads to a concomitant increase in displacements in the SH3 domain such
that the interactions between the SH3 domain and the N-terminal lobe of the kinase
become less well defined. When the tail is released from the SH2 domain, the fluctuations
of the SH3 domains in respect to the N-terminal lobe of the kinase are seen to increase
substantially. It was also observed that when the tail is released from the SH2 domain, the
fluctuations of the SH3 domain with respect to the N-terminal lobe of the kinase seem to
increase substantially.
Further studies indicated that the free rotation of the SH2 and the SH3 domains is
due to the disruption of the hydrogen bonded structure of the connector by water
molecules. Thus, it is presumed that the engagement of the SH2 and SH3 domains by their
internal docking sites keeps the hydrogen bonds in the connector locked in place and
makes the two domains rigid.
The maintenance of the relatively rigid structure is crucial for the ability of the
connector to couple dynamical fluctuations between the SH2 and the SH3 domains. This
was proved by replacing some residues in the c-Src connector by glycine, which had a
dynamic effect on the dynamics of the protein. The introduction of glycine made the SH2
and the SH3 domains become significantly less correlated in their motions even though
the phosphorylated tail is connected tot eh SH2 domain. The introduction of flexibility
into the SH2 and the SH3 connector in the simulation therefore mimics the effect of tail
dephosphorylation in the simulations.
In the study conducted by Ma and Huang et al, Src was found to be an effector of
G proteins. Previously, Src has been shown to have a role in the signal transduction of Gprotein coupled receptors. In this study the authors showed that Gs and GI stimulate
the kinase activity of c-Src using kinase assays and immunoprecipitation studies.
Mutation
experiments
and
a
trypsin
digestion
protection
assay
and
co-
immunopreciptation studies helped to determine that these two G proteins bind to the
catalytic domain of Src and change it’s conformation leaving the active site assessable to
substrates. The activation of GPCRs has been shown to increase the activity of Src family
kinases but the mechanisms were not clear until this paper demonstrated that Gs and
GI stimulate the kinase activity of Src and Hck (another Src family kinase) (Ma and
Huang et al 2000).
Previously it has been shown that c-Src kinase activity can be regulated by
tyrosine phosphorylation of T-527, and that this is inhibitory to the activity of the
molecule. Src is active when T-527 is in the dephosphorylated state. The study by Ma
and Huang found no effect of G-proteins on this process in contrast to the simulation
studies done by Young et al at Rockefeller University . It has also been shown that
autophosphorylation of tyrosine 416 at the activation loop leads to full activate of Src
family kinases. In the inactivated form of Src, the molecule is in a -helical conformation
and when activate the loop becomes extended. The phosphorylation of T-416 might
stabilize this extended conformation. In this study Gs and GI increases the
autophosphorylation of c-Src at tyrosine 416 (Ma and Huang et al 2000).
As stated previously, c-Src kinase activity can be modulated by either tyrosine
phosphorylation or conformational change. Ma and Huang showed that the G-proteins
interact with the catalytic domain of Src and this suggests that a conformational change
model is required for kinase activity in this experiment. Enzyme kinetic experiments
showed that the G proteins decreased the Km for the peptide substrate. This might
suggest that G protein binding changes the conformation of the Src molecule and allows
the peptide access to the active site. In other studies involving Src it was shown that the
activation loop of c-Src forms and -helix that blocks that peptide-binding site of the
catalytic domain. When the molecule is in it’s active state, the activation loop moves
away from the entrance of the catalytic cleft and allows substrates to bind the active site
(Ma and Huang et al taken from Yamaguchi et al). The authors of this study propose that
the Gs and GI binding to the catalytic domain controls the activation loop. They
believe could relieve steric hindrance at the entrance to the catalytic cleft and increased
accessability to the active site for substrates. Since the Tyrosine 416 is hidden in the
catalytic cleft in the inactive form, the conformational change initiated by the G proteins
would also make this side chain more exposed for autophosphorylation. All these aspects
would lead to increased kinase activity in response to the Gs and GI proteins (Ma and
Huang et al 2000).
References:
1. Thomas and Brugge, " Cellular functions regulated by src family kinases”, 1997,
Annual review of cell and developmental biology 13: 513-609
2. Superti-Furga Team: Regulation of the Src and Abl protein tyrosine kinases and their
cytoplasmic and nuclear phosphorylation circuits. Online at http://www.emblheidelberg.de/emblGroup/researchReport/rr00_59.pdf
3. Engen J. “Src-family proteins”. Online. http://www.hxms.com/je/srcfam.html
4. Schindler T, et al., “Crystal Structure of Hck in Complex with a Src Family-Selective
Tyrosine Kinase Inhibitor” May 1999 Mol Cell 3: 639-648.
5. Xu W.Q. editor et al., “Hot Papers in Crystal Structure” The Scientist 13: 10 May
1999.
6. Young M et al., “Dynamic Coupling between the SH2 and SH3 Domains of c-Src and
Hck Underlies their Inactivation by C-terminal Tyrosine Phophorylation April 2001 Cell
105: 115-126.
7. Ma Yong et al. "Src Tyrosine Kinase is a Novel Direct Effector of G Proteins" Sept
2001 Cell 102: 635-646.
8. Martin, Steven G. "The hunting of the Src" Online
http://www.nature.reviews.molecular cell biology