Tyrosine
kinase Hck
Sarah Cook '11 and Sally Moseley '12
Contents:
I.
Introduction
Tyrosine
phosphorylation has roles
in cell proliferation, migration, differentiation, and survival (Thomas
et al.
1997).
A family of kinases that participate in tyrosine phosphorylation
is the
Src kinase family.
Members of this
kinase family phosphorylate a variety of substrates involved in many
cellular
processes.
The Src kinases’
ability to
act on many different
substrates allows for the diversity of effects
that
result from their phosphorylase activity.
This
Src kinase family was first discovered
with the finding that a member of this familiy, v-src,
is implicated in Rous sarcoma virus. This
viral form of src
leads to rapid
tyrosine phosphorylation in the cytoplasm of host cells,
making it a
powerful
cellular transforming agent.
Therefore,
a greater understanding of these kinases is important for understanding
the
signal transduction pathways involved in human viruses.
All
Src kinase family enzymes are
composed of 3 conserved domains and kinase-specific linker between 2 of
the three
domains.
This linker allows for
interactions
specific to each kinase’s function. Here, a member of this
family, tyrosine
kinase Hck, is reviewed.
Hck was
crystallized as a dimer
.
Hck is found in
lymphoid and myeloid cells bound to B-cell receptors in inactive B
cells.
A lack of Hck results in
developmental
defects and suppressed immunity.
II.
General Structure
The
tyrosine kinase Hck is
composed
of four parts: the catalytic
domain,
the SH2
domain, the SH3
domain, and the linker
.
The catalytic
domain is composed
of a N-lobe
and a C-lobe
,
which interact with phosphate groups and
other
substrates, respectively, and is responsible for tyrosine kinase
activity.
The N-lobe
is composed of
mostly β sheets, allowing for
its interactions with incoming phosphate
groups
.
The C-lobe
is composed
of mostly
α-helicies, which bind substrates in the substrate binding
groove
.
The SH2
and SH3
domains
are bound to the
catalytic domain on the side opposite its active site and provide
stability and
regulation of the catalytic domain
.
The
SH2
domain is made up
of 3 major β sheets and 2 α helicies
. SH2
is bound to the C- lobe
of the catalytic domain via a
phosphotyrosine
residue (residue 527) and Pro 531 on
the C-lobe
.
Because there are three residues between the
phosphotyrosine residue and Pro 531, the peptide bends away from the SH2
domain (link),
creating a lower-affinity interaction
.
SH2
is also connected to
the N-lobe
of the catalytic domain by a
polyproline type II (PPII) helix that spans from residues
Pro 244 to
Trp
254
.
The
hydrophobic surface of
the third domain SH3
is created by 5 β sheets
, which allows it to bind
to the
SH2-kinase linker.
The
hydrophobic surface of the
third domain SH3
binds to this SH2-kinase
linker
.
This linker
gives Hck much of its protein
specificity because the sequence of the linker is highly variable
amongst Src
kinases.
III.
Phosphorylation
Kinase
activity is correlated to the conformation of the protein. The 'closed'
conformation is the active form because the two lobes of the catalytic
site bring the phosphate group and substrate into closer proximity.
The
'open' conformation is the inactive form because the lobes are bent
away from each other, disallowing interactions between the phosphate
group and substrate.
Hck's
conformation as either 'closed' or 'open' is
dependent
on autophosphorylation at Tyr 416.
When unphosphorylated, Glu 310 on an α-helix, αC,
of the N-lobe of the catalytic
domain
forms a hydrogen
bond with Arg 385
,
causing Glu 310 to be
flipped outward. In this
inactive state, SH3 is bound to the SH2-kinase linker via residue Trp
260 on SH3
and αC
on the N-lobe of the catalytic
domain
.
Alternately,
when Tyr 416 in the activation segment of the catalytic domain is
phosphorylated, the phosphorylated tyrosine interacts with Arg 385
,
barring the Glu 310-Arg 385 interaction. In this conformation, Glu 310
and αC are able to rotate inward, causing the release of SH3
from the SH2-kinase linker. The flexible linker allows the enzyme to
adopt the active conformation.
IV.
Comparison to PKA
The
structure of tyrosine kinase Hck has many similarities to that of
cyclic-AMP-dependent protein kinase A (PKA) complexed with ATP
. The
active sites of Hck and ATP-bound PKA have
very similar conformations and superimposition of the two kinases shows
only a
3° difference
in the angles between the N-
and C-lobes (link).
This similarity to active ATP-bound PKA is
important
because it shows that Hck is also in its active conformation. The
N-
lobe is slightly further from
the C-
lobe in Hck, overlapping the
normal position αC in PKA.
Due
to this slight difference in configuration, some residues on αC
in each enzyme are facing different
directions (link).
In
PKA, the Glu 91 residue,
equivalent to Glu 310 in Hck, faces inward forming an ionic bond with
Lys 72
,
which directs the α- and β-phosphates
of
ATP.
The charged Glu 91
adjacent to Lys 72
plays a
large role in PKA’s enzymatic activity.
Hck deals with having an
outward facing Glu 310 by the
phosphorylation-dependent conformation change described in the above
section
.
This allows
for a unique allosteric
regulation of Hck.
V.
Applications
Over-activation of Hck may
lead to
stunted cell growth, as evidenced by its association with unstimulated
B-cells
(Sicheri et al.,
1997) and suppressed growth of mutated yeast cells with
Src expression (Trible et al.,
2006). This could be part of the
mechanism by which oncoviruses disrupt cell activity. Nef, an
HIV-1
protein, increases viral pathogenicity by activating Src family kinases
that
initialize a signal transduction pathway (Trible et
al., 2006).
Therefore, Hck is also
claimed to be
an essential factor in the progression of the disease to acquired
immune
deficiency syndrome (AIDS) (Choi et
al., 2004). In the
absence of
Nef, autophosphorylation of Tyr 416 is a slow process involving the
subunits
SH2
and SH3
and ATP. The two subunits SH3
and the catalytic domain
continue to associate with each other. In the presence of
Nef, however,
SH3
binds to Nef and is completely detached from the catalytic domain and
only
associated with the SH2.
This leaves Hck in an extended, open
conformation that allows rapid phosphorylation of Tyr 416 and thus
maximizes
the activation of the kinase. It is possible that
Hck’s enhanced activity
in the presence of Nef in HIV-infected people may have something to do
with
Hck’s correlation with unstimulated B cells. If
hightened Hck activity
causes the inactivity of B cells, the suppression of the immune system
that
comes with HIV could be due to inactive B cells.
VI.
References
Choi,
Hyun-Jung
and Thomas E. Smithgall. 2004 Conserved Residues in the HIV-1
Nef Hydrophobic Pocket are
Essential for Recruitment
and Activation of the Hck
Tyrosine
Kinase. Journal of Molecular
Biology 5:1255-1268
Sicheri,
Frank, Ismail Moarefi, and John Kuriyan. 1997. Crystal
structure of the Src family tyrosine kinase Hck. Nature 385: 602-609.
Thomas,
S. M. and J. S.
Brugge.
1997. Cellular Functions Regulated by SRC Family Kinases. Annu. Rev.
Cell Dev.
Biol. 13: 513-609.
Trible,
Ronald P., Lory Emert-Sedlak, and Thomas E. Smithgall. 2006.
HIV-1 Nef selectively activates Src family kinases Hck, Lyn,
and
c-Src through direct SH3 domain interaction. Journal of Biological
Chemistry 281: 27029-38.
Zheng,
Jianhua,
Elzbieta A. Trafny, Daniel R. Knighton, Nguyen-Huu Xuong, Susan S.
Taylor, Lynn F. Ten Eyck, and Janusz M. Sowadski. 1992. A
refined
crystal structure of the catalytic subunit of cAMP-dependent protein
kinase complexed with MnATP and a peptide inhibitor. Acta Cryst. 49:
362-365.
Back
to Top