(Received for publication, August 10, 1995; and in revised form, October 5, 1995)
From the
Lyn is a member of the Src family of protein-tyrosine kinases
that can readily undergo autophosphorylation in vitro. The
site of autophosphorylation is Tyr which corresponds to
the consensus autophosphorylation site of other Src family tyrosine
kinases. The rate of autophosphorylation is concentration-dependent,
indicating that the reaction follows an intermolecular mechanism.
Autophosphorylation results in a 17-fold increase in protein-tyrosine
kinase activity. Kinetic analysis demonstrates that phosphorylation of
a substrate peptide by Lyn following autophosphorylation occurs with a
63-fold decrease in K
but no significant
change in V
, suggesting that autophosphorylation
relieves the conformational constraint that prevents binding of the
substrate peptide to the active site of the kinase. Using a
phosphotyrosine-containing peptide (pYEEI) that has previously been
shown to bind to the Src homology 2 (SH2) domain of Src family tyrosine
kinases with high affinity, we found that autophosphorylation results
in a significant decrease in accessibility of the Lyn SH2 domain,
indicating that conformational changes in the protein kinase domain
induced by autophosphorylation can be propagated to the SH2 domain. Our
study suggests that autophosphorylation plays an important role in
regulating Lyn by modulating both its kinase activity and its
interaction with other phosphotyrosine-containing molecules.
The protein products of the Src family of oncogenes and proto-oncogenes are non-receptor protein-tyrosine kinases that are believed to play important roles in controlling the growth, proliferation, and differentiation of many cell types (see (1) for review). Src family kinases are highly homologous in structure; they all contain an N-terminal myristoylation domain, a unique domain, Src homology 2 (SH2) and Src homology 3 (SH3) domains, a protein kinase domain, and a C-terminal regulatory domain. Studies of transforming mutants of several Src family kinases have provided evidence that interactions between these domains are important in the regulation of kinase activity. Two major consensus tyrosine phosphorylation sites have been identified in Src family kinases: (i) the autophosphorylation site in the protein kinase domain and (ii) the tyrosine phosphorylation site in the C-terminal regulatory domain. Autophosphorylation correlates with activation of the kinases, while phosphorylation of the C-terminal regulatory tyrosine suppresses kinase activity (see (1) for review). The C-terminal Src kinase has been shown to phosphorylate several members of this family and is thought to play a critical role in regulating their activity(2) .
Phosphorylation of the C-terminal tyrosine in
Src family kinases negatively regulates their activity. Several lines
of evidence suggest that this regulation is governed by an
intramolecular interaction between the phosphorylated C-terminal
tyrosine and sequences in the SH2 domain that somehow stabilizes the
inactive conformation of the kinases (see (1) and (3) for review). In addition to binding to the C-terminal
regulatory phosphotyrosine, the SH2 domain has also been shown to bind
other phosphotyrosine-containing proteins(4) . Interaction with
exogenous phosphotyrosine-containing proteins is thought to play an
important role in the cellular functions of the kinases (see (4) and (5) for review). Using combinatorial peptide
libraries, structural determinants in phosphotyrosine-containing
proteins necessary for high affinity binding to the SH2 domain of Src
family kinases have been determined(6) . Peptides displaying
high affinity binding to the SH2 domain of Src family kinases
invariably contain a phosphotyrosine followed C-terminally by two
acidic amino acids and then a hydrophobic residue. A phosphopeptide,
pYEEI, derived from the hamster polyoma virus middle T antigen contains
all the structural features important for high affinity binding to SH2
domains of Src family kinases(7, 8) . The structural
basis of the high affinity interaction between the phosphopeptide and
the SH2 domain was elucidated from the crystal structure of the
pYEEI-Src SH2 domain complex(9) . The crystal structure reveals
two major binding pockets for pYEEI, one for the phosphotyrosine and
the other for the more C-terminal Ile residue. The
phosphotyrosine-binding pocket contains an Arg residue that forms
hydrogen bonds with the phosphate moiety and two basic residues that
bind to the aromatic ring of the phosphotyrosine through amino-aromatic
interactions. The Ile-binding pocket contains several hydrophobic
residues responsible for hydrophobic interactions with the Ile residue.
In addition to the two binding pockets, electrostatic interactions
between the two Glu residues of the peptide and several basic residues
of the SH2 domain also contribute to the high affinity binding of the
peptide in the SH2 domain of pp60(9) .
Studies on the role of autophosphorylation in the regulation of the
biological activity of products of the c-src and v-src genes, pp60 and pp60
,
respectively, are well documented (see (1) for review).
Mutation of the autophosphorylation site (Tyr
) of
pp60
generates a mutant displaying somewhat reduced
kinase activity but maintaining full oncogenicity(10) . Thus,
autophosphorylation does not seem to play a very significant role in
regulating the kinase activity and oncogenicity of pp60
because it is not obligatory for kinase activity or transforming
potential. A similar conclusion on the kinase activity of
pp60
can be made because nonphosphorylated
pp60
displays significant kinase activity, and
autophosphorylation leads to a 1.5-2-fold increase only in its
kinase activity(36) . This is in contrast to insulin- and
epidermal growth factor receptors which require autophosphorylation,
not only for kinase activity, but also for normal biological responses.
No detailed analyses of the effect of autophosphorylation on the
regulation of the kinase activity and cellular functions of other
members of the Src family have been documented.
We reported
previously the purification of a Src family tyrosine kinase, Lyn, from
extracts of bovine spleen(13) . Autophosphorylated Lyn was
shown to be highly efficient in specifically phosphorylating a peptide
cdc2(6-20) which is derived from the cell cycle control kinase
p34(14) . Lyn is thought to play an important
role in relaying signals originating from a number of hematopoietic
cell surface receptors such as the B-cell
receptor(15, 31, 32, 33, 34) .
Stimulation of B-lymphocytes, by engagement of the B-cell receptor,
results in rapid tyrosine phosphorylation and activation of Lyn (see
Refs. 15, 16, and 35 for review). How stimulation of the B-cell
receptor regulates Lyn activity is not fully understood. Likewise, it
is unclear how interplay between the SH2 domain, the
autophosphorylation site, and the C-terminal regulatory domain of Lyn
modulates its kinase activity and its ability to relay signals from
cell surface receptors.
Herein we have used detailed biochemical analyses to investigate the mechanism of autophosphorylation and the effect of autophosphorylation on the protein kinase activity of Lyn. Using the pYEEI peptide, we have also studied the effect of autophosphorylation on the conformation of Lyn by measuring the accessibility of its SH2 domain to the phosphopeptide. Our studies demonstrate that autophosphorylation plays a significant role in regulating the kinase activity of Lyn and show that structural changes induced by autophosphorylation can be transmitted to the SH2 domain.
Figure 1:
Characterization of the purified
recombinant Lyn preparation. a, SDS-PAGE analysis followed by
Coomassie Blue staining of 1.5% of the total purified Lyn preparation.
Lyn was autophosphorylated to a stoichiometry of 1 mol of
PO incorporated per mol of kinase, and
samples containing equal amounts of protein either before (-ATP)
or after (+ATP) autophosphorylation were subject to immunoblot
analysis using anti-Lyn antibody (b) or anti-phosphotyrosine
antibody (PY69) (c).
To monitor the change in Lyn tyrosine
kinase activity, autophosphorylation of the kinase was carried out
under identical conditions as described above. At timed intervals, 5
µl of the reaction mixture was added to a mixture containing 45
µl of kinase assay buffer,
[Lys]cdc2(6-20) peptide, and
[
-
P]ATP to initiate phosphorylation of the
[Lys
]cdc2(6-20) peptide. The peptide
phosphorylation was allowed to proceed at 30 °C for 5 min only to
avoid any significant further autophosphorylation of Lyn. The assay
conditions were the same as those detailed under Protein Kinase
Assay.
For proteolytic
digestion, the reaction mixture was first dialyzed against 2 2
liters of dialysis buffer overnight to remove free ATP. The dialyzed
sample was concentrated and then alkylated by treatment with
4-vinylpyridine (2 µl per 100 µl of concentrated mixture) at 37
°C for 3 h. After addition of 30 µg of BSA as carrier protein,
the autophosphorylated Lyn was precipitated by incubating with 0.9 ml
of ethanol at -20 °C overnight and then washed with 1 ml of
ethanol at -20 °C. After removal of the residual ethanol by a
Speed Vac, the precipitated proteins were exhaustively digested with
tosylphenylalanyl chloromethyl ketone-treated trypsin (1 mg/ml) in a
volume of 150 µl at 37 °C for 48 h. The tryptic phosphopeptide
fragment was isolated by reverse phase HPLC before analysis by the
two-dimensional thin layer electrophoresis-thin layer chromatography
(TLC) procedure.
For phosphopeptide mapping, each of the samples
(the phospho-lyn(391-400), the tryptic phosphopeptide fragment
derived from autophosphorylated Lyn, and a mixture of both) was applied
to a TLC plate. The first dimension was thin layer electrophoresis in a
pH 3.5 buffer (pyridine, acetic acid, and HO in a ratio of
1:10:89) at 500 V for 2.5 h, and the second dimension was TLC in a
buffer containing 1-butanol, pyridine, acetic acid, and H
O
in a ratio of 15:10:3:12. The radioactive spot on the TLC plate was
located by autoradiography.
In same of the recombinant Lyn preparations, a protein band corresponding to a 54-55-kDa form of Lyn also exists (Fig. 7c). This protein band does not cross-react with the anti-phosphotyrosine antibody, indicating that it is not tyrosine-phosphorylated. However, upon incubation with potato acid phosphatase, this 54-55-kDa form of Lyn disappears, suggesting that it represents a serine/threonine-phosphorylated form of Lyn (data not shown).
Figure 7: Time course of changes in stoichiometry of autophosphorylation, tyrosine kinase activity, and accessibility of the SH2 domain of Lyn to the immobilized pYEEI peptide. a, stoichiometric analysis of Lyn autophosphorylated for the times indicated. b, kinase activity of Lyn autophosphorylated for the times indicated. c, SDS-PAGE followed by immunoblotting of Lyn bound to the immobilized pYEEI peptide using the anti-Lyn antibody. Lyn was autophosphorylated for 0, 1, 15, 30, or 60 min. d, densitometric analysis of immunoblot shown in c.
Figure 2:
Time course of autophosphorylation and
change in tyrosine kinase activity of Lyn. Purified Lyn was
autophosphorylated by incubation with
[-
P]ATP. At designated time intervals,
aliquots of the mixture were removed for SDS-PAGE analysis (a), determination of stoichiometry of autophosphorylation (b), and protein-tyrosine kinase activity using
[Lys
]cdc2(6-20) peptide as the substrate (c).
Figure 3: Effect of Lyn concentration on the rate of autophosphorylation.
The initial velocity of autophosphorylation was
determined using 160 nM Lyn and various concentrations of ATP.
Lineweaver-Burke analysis of the data shows that the K for ATP is 14 µM and the V
of
autophosphorylation at 160 nM Lyn is 1.5
10
µmol/min/mg. Our data indicate that the slow in vitro rate of autophosphorylation is a result of the very
low catalytic efficiency of trans-autophosphorylation once
MgATP has bound to the active site. As we have shown that Lyn
autophosphorylation is concentration-dependent, a higher V
value would have been attained if a higher Lyn
concentration had been used in the assay.
It would be informative to
know the K and V
values for
Lyn in the autophosphorylation reaction. However, since Lyn acts as
both the enzyme and the substrate in the reaction, it is not possible
to determine these kinetic parameters by conventional kinetic analysis.
Figure 4: Determination of the autophosphorylation site of Lyn by tryptic phosphopeptide mapping. Autoradiographs of tryptic phosphopeptide maps generated with autophosphorylated Lyn (a), phosphorylated lyn(391-400) peptide standard (b), and a mixture of both the Lyn tryptic phosphopeptide and the phosphopeptide standard (c).
The sequence of
Lyn around tyrosine 397 is highly homologous to the sequence around the
known autophosphorylation site in pp60 (Fig. 5),
strongly suggesting that Tyr
is the autophosphorylation
site in Lyn. A peptide derived from residues 391-400 of Lyn,
lyn(391-400), was synthesized and radioactively phosphorylated.
The resulting phosphopeptide VIEDNE(pY)TAR was used as the
phosphopeptide standard for identification of the Lyn
autophosphorylation site. Both the standard and the tryptic
phosphopeptide fragment derived from Lyn migrated to an identical
position in the two-dimensional phosphopeptide maps (Fig. 4, b and c), confirming that Tyr
is indeed
the autophosphorylation site.
Figure 5:
Homology of the amino acid sequences
flanking the autophosphorylation site of insulin receptor (IRK), pp60, and Lyn. Amino acid sequences
surrounding the autophosphorylation sites of IRK (Tyr
),
pp60
(Tyr
), and Lyn (Tyr
).
Autophosphorylation sites are marked by asterisks, and the
highly conserved arginine residue (Arg
of IRK,
Arg
of pp60
, and Arg
of
Lyn) in the catalytic loop is marked by a solid circle. The arrows mark the peptide fragments containing Tyr
of Lyn that can be generated by exhaustive tryptic
digestion.
Figure 6: Specificity of binding of Lyn to the immobilized pYEEI peptide. a, Lyn was preincubated with various competing agents prior to incubation with immobilized pYEEI peptide. Proteins bound to the immobilized pYEEI peptide were eluted and separated by SDS-PAGE followed by immunoblot analysis with anti-Lyn antibody. Lane 1, Lyn bound to the immobilized pYEEI peptide in the absence of any competing agents. The competing agents used were free pYEEI peptide (lanes 2-5), YEEI peptide (lanes 6-8), phosphotyrosine (lanes 9 and 10), unrelated nonspecific peptide 1 (lanes 11 and 12), and unrelated nonspecific peptide 2 (lanes 13 and 14) at the concentration indicated. b, densitometric analysis of immunoblot shown in a. The relative amount of Lyn bound to the immobilized pYEEI peptide in the presence of the indicated competing agents is expressed as densitometry units.
As mentioned under Characterization of Purified Recombinant Mouse Lyn, some of our purified Lyn preparations contain a 54-55-kDa form of Lyn which might arise as a result of serine/threonine phosphorylation in vivo. As indicated in Fig. 7, the 53-kDa, 54-55-kDa, and 56-kDa forms of Lyn can undergo autophosphorylation and the autophosphorylation-induced decrease in SH2 domain accessibility.
In the present study we have demonstrated that Lyn
autophosphorylation correlates with an increase in its kinase activity.
Our observations support the notion that autophosphorylation stabilizes
the active conformation of the kinase and thereby leads to activation
of the kinase. Autophosphorylation occurs exclusively at
Tyr. This agrees well with similar studies on other
members of the Src family (see (1) for review). We have shown
that Lyn autophosphorylation follows an intermolecular or trans-mechanism. Similar to Lyn, autophosphorylation of the
insulin receptor which occurs by a trans-mechanism is a
prerequisite for its activation(11) . Comparison of the
sequence surrounding the autophosphorylation site in the insulin
receptor (Tyr
) with that of Lyn reveals significant
homology (Fig. 5)(1, 11) . The similar
enzymatic properties of these two kinases in addition to the sequence
homology surrounding their autophosphorylation sites suggest that the
two kinases follow similar molecular mechanisms of autophosphorylation
and autoactivation.
Based upon the crystal structure of the insulin
receptor tyrosine kinase domain (IRK), a model explaining how trans-autophosphorylation leads to activation of the insulin
receptor has been postulated(11) . In this model, the
nonphosphorylated IRK is locked in an inactive conformation in which
the ``self''-autophosphorylation site (Tyr) is
``engaged'' in the substrate-binding region of the active
site. Upon autophosphorylation, the phosphate moiety of Tyr(P)
of IRK is believed to electrostatically interact with
Arg
in the catalytic loop. Presumably, such an
interaction stabilizes the active conformation by
``disengaging'' Tyr(P)
from the
substrate-binding region in the active site. Such a model can also be
used to explain the kinetic consequences of Lyn autophosphorylation.
The ``self'' Tyr
of Lyn blocks the binding of
substrate, and, as a result, nonphosphorylated Lyn is in an inactive
conformation. This model is supported by the fact that the K
value of nonphosphorylated Lyn for the exogenous
peptide substrate is extremely high (25 mM). Presumably, upon
autophosphorylation, Tyr(P)
is ``disengaged''
from the substrate protein-binding region in the active site. As a
result, binding of substrate to the active site is allowed and the
kinase is activated. This model is further substantiated by the 63-fold
decrease in K
of Lyn for the substrate peptide
after autophosphorylation. We postulate that Arg
in the
catalytic loop, homologous to Arg
of IRK, is the basic
residue binding to Tyr(P)
and in turn allowing binding of
the substrate peptide to Lyn by disengaging Tyr(P)
from
the substrate-binding regions in the active site (Fig. 5).
Confirmation of the putative Tyr(P)
-Arg
interaction requires the elucidation of the crystal structure of
autophosphorylated Lyn.
Similar studies of pp60 shows that autophosphorylation led to a 2-fold increase in kinase
activity while our study reports a 17-fold increase in the kinase
activity of Lyn upon autophosphorylation(36) .
Autophosphorylation of pp60
did not alter the K
value but caused a 2-fold increase in V
for its substrate protein,
casein(36) . This is in sharp contrast to Lyn where
autophosphorylation alter the K
but not the V
for its substrate peptide. Thus, despite the
high degree of sequence homology between the protein kinase domains of
pp60
and Lyn, the extent and molecular mechanisms of
autoactivation of these two kinases are quite different.
Previous
studies have shown that Lyn is physically associated with a number of
hematopoietic cell surface receptors including the B-cell
receptor(15) , Fc-receptor I(31) , Fc-
receptor I(32) , interleukin 7 receptor(33) , and
granulocyte colony-stimulating factor receptor(34) .
Stimulation of these cell surface receptors results in rapid
phosphorylation and activation of Lyn in vivo (see Refs. 15,
16, and 36 for review), and it is therefore intriguing that the in
vitro autophosphorylation of Lyn occurs at a very slow rate. It is
possible that the physical association of Lyn with these receptors
increases the effective concentration of Lyn available for
autophosphorylation. Likewise, upon stimulation of the receptors,
conformational changes originating from the receptors could be
propagated to the kinase domain of Lyn and somehow render the active
site more accessible to the trans-Tyr
residue of
the neighboring Lyn molecule.
There is a substantial body of
evidence supporting the involvement of Src homology 2 (SH2) domains in
the regulation of the activity of Src family kinases. The generally
accepted model involves binding of the C-terminal phosphotyrosine
(Tyr of Lyn and Tyr
of
pp60
) to the SH2 domain which forces the kinase to
assume an inactive conformation (29) ). Upon dephosphorylation
of the C-terminal phosphotyrosine, its interaction with the SH2 domain
is disrupted and this allows the kinase to assume the de-repressed
conformation and undergo autophosphorylation (Fig. 8). The
detailed structural basis for inactivation by such an interaction is
not understood. In addition to the C-terminal phosphotyrosine-SH2
domain interaction, interaction between SH2 domains of Src family
kinases with exogenous phosphotyrosine-containing proteins has been
documented (see Refs. 15, 16, and 35 for review). Interaction of the
SH2 domain of pp60
with the exogenous pYEEI peptide
inhibits its kinase activity. Furthermore, photoaffinity cross-linking
of the SH2 domain of pp60
with a pYEEI peptide analog
partially inactivated the kinase(26) . Based upon these
observations, Garcia et al.(26) postulated that
occupancy of the SH2 domain induces a conformational change that is
transmitted to the kinase domain and attenuates the tyrosine kinase
activity of pp60
(26) .
Figure 8:
A model depicting the three conformational
states of Lyn. Lyn can exist in at least three hypothetical
conformations. In the inactive conformation (conformation 1),
Tyr is phosphorylated, presumably by C-terminal Src
kinase or a related kinase. Intramolecular interaction between the
Tyr(P)
and the SH2 domain leads to inactivation of Lyn.
Dephosphorylation of Lyn in conformation 1 by an as yet unknown
phosphatase converts Lyn to a fully nonphosphorylated state (conformation 2). Autophosphorylation of Lyn at Tyr
gives rise to a fully activated tyrosine kinase (conformation
3).
Our observation that
autophosphorylation of Lyn leads to a decrease in the accessibility of
its SH2 domain to the immobilized pYEEI peptide provides evidence for
the propagation of conformational changes from the kinase domain to the
SH2 domain of Lyn. How would the propagation of conformational changes
occur and what is the structural basis dictating the functional
interaction between the kinase domain and the SH2 domain of Lyn? Using
the crystal structure of the catalytic subunit of cAMP-dependent
protein kinase as the template, Veron et al.(27) revealed a putative helix motif (the A-helix motif)
in Src family kinases by homology modelling; this A-helix motif forms
the basis of a hypothetical model for the cross-talk between the kinase
domain and the SH2 domain documented for pp60 and Lyn.
In this model, the A-helix motif serves as a linker between the
catalytic core and the SH2 domain of Src family kinases(27) .
This hypothetical A-helix motif can potentially interact with essential
amino acid residues in the catalytic loop as well as residues in close
vicinity to the autophosphorylation site. Presumably, these
interactions allow propagation of the autophosphorylation-induced
conformational changes from the protein kinase domain through the
A-helix motif to the SH2 domain which may account for the decreased SH2
domain accessibility of autophosphorylated Lyn to the pYEEI peptide (Fig. 6).
GTPase activator protein, mitogen-activated protein
kinase (MAP kinase), and phospholipase C- are among the proteins
that are rapidly tyrosine-phosphorylated following stimulation of the
B-cell receptor (see (15) for review). These proteins are
believed to play essential roles in transducing signals initiated by
stimulation of the B-cell receptor(37) . When phosphorylated,
these phosphoproteins can bind to the SH2 domain of Lyn, thereby
providing a means of transmitting signals via Lyn that have initiated
from the B-cell receptor. The binding site for these phosphoproteins
has been mapped to the unique and SH2 domain of Lyn(37) . Our
observation that autophosphorylation decreases the accessibility of the
SH2 domain of Lyn to the pYEEI peptide suggests that
autophosphorylation may modulate binding of Lyn to
phosphotyrosine-containing molecules in vivo.
From our
data, we postulate that Lyn exists in at least three hypothetical
conformational states in vivo (Fig. 8). The inactive
form of Lyn is represented by conformation 1. In this conformation, the
C-terminal regulatory tyrosine is phosphorylated, presumably by
C-terminal Src kinase or a related kinase (38) . ()Interaction between the C-terminal phosphotyrosine and the
SH2 domain suppresses the kinase activity and prevents its SH2 domain
from binding to exogenous tyrosine-phosphorylated protein molecules.
Dephosphorylation of the C-terminal phosphotyrosine by an as yet
unidentified phosphatase allows the kinase to assume a derepressed
conformation which displays low or no kinase activity (conformation 2).
Owing to its ability to bind the pYEEI peptide, Lyn in this
conformation can potentially bind tyrosine-phosphorylated proteins. The
kinase can then be activated by autophosphorylation giving rise to the
fully active form of the enzyme (conformation 3). In this conformation,
Lyn is capable of phosphorylating its protein substrates but our data
suggest that its accessibility to phosphotyrosine-containing proteins
is greatly reduced.
Since our observation suggests that
autophosphorylation is obligatory for autoactivation of Lyn,
dephosphorylation of Tyr(P) must be an essential step in
deactivation of the kinase. The protein-tyrosine phosphatase
responsible for dephosphorylation of Tyr(P)
of Lyn has
not been identified. Identification of this phosphatase will be
important for understanding how the activity of this enzyme is
regulated.