(Journal of Leukocyte Biology. 2000;68:603-613.)
© 2000
by Society for Leukocyte Biology
Src kinase-mediated signaling in leukocytes
eljka Korade-Mirnics and
Seth J. Corey
Department of Pediatrics and Pharmacology, University of Pittsburgh School of Medicine, Pennsylvania
Correspondence: Seth J. Corey, M.D., Division of Hematology-Oncology, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213. E-mail: scorey{at}pitt.edu
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ABSTRACT
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A concert of antigens, antibodies, cytokines, adhesion molecules, lipid
factors, and their different receptors mediate leukocyte development
and inflammatory responses. Regardless of the stimulus and receptor
type, members of the Src family of protein tyrosine kinases (PTKs) play
a critical role in initiating the numerous intracellular signaling
pathways. Recruited and activated by the receptor, these Src PTKs
amplify and diversify the signal. Multiple pathways arise, which affect
cell migration, adhesion, phagocytosis, cell cycle, and cell survival.
Essential nonredundant properties of Src PTKs have been identified
through the use of gene targeting in mice or in the somatic cell line
DT40. Because of their role in mediating leukocyte proliferation and
activation, Src PTKs serve as excellent drug targets. Inhibitors of Src
family members and dependent pathways may be useful in the treatment of
human diseases similar to drugs known to inhibit other signal
transduction pathways.
Key Words: signal transduction • leukocytes
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LEUKOCYTE SIGNAL TRANSDUCTION
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Lymphocytes and phagocytes protect the body against infection and
excessive inflammation. To perform these functions, leukocytes must
sense infection or inflammation, migrate to and anchor at the involved
site, ingest and destroy the infectious agent, and clean up the
detritus. No single type of leukocyte can perform all of these
functions. Different leukocyte cells must interact and coordinate their
activities with other types and accessories such as endothelial cells
and fibroblasts. Peptides in the form of antibodies and cytokines,
lipid mediators, extracellular matrix, and reactive oxygen species
provide signals for this concert of infection control and inflammation.
Receptors, primarily on the cell surface of leukocytes, receive these
signals and process them via second messengers or cascades of pathways.
As a result of this process of signal transduction, neutrophils,
eosinophils, monocytes/macrophages, T cell lymphocytes, or B cell
lymphocytes produce inflammation.
Leukocytes develop, differentiate, and expand by growth factors and
cytokines operating on their cognate receptors, which either contain
intrinsic enzymatic activity or recruit an enzyme(s) from the cytosol.
They sense chemoattractant gradients via seven-transmembrane, G
protein-coupled receptors (GPCR). Leukocytes attach to other cells or
extracellular matrix via the integrins. Antigen is presented or
antibody is retained by leukocytes via multimeric receptors. Typically
present on any particular leukocyte, five different classes of
receptors may be sequentially or concurrently activated
[1
2
3
4
5
6
]. One more common theme is that each receptor
class utilizes a member of the Src family of protein tyrosine kinases
(PTKs) as a secondary effector (Fig. 1
and Table 1
).
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Figure 1. Src Participates in the signal transduction through different receptor
types. There are five different types of receptors: receptors with
encoded effector domains (RTK), G protein-coupled receptors (GPCR),
receptors for adhesion molecules (integrins), hematopoietin/cytokine
receptors (H/CR), and multimeric receptors. Effector enzymes commonly
use serine/threonine or tyrosine kinases. TK, tyrosine kinase domain is
shown for the RTK.
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Src family members are non-receptor PTKs, which are expressed in a
variety of hematopoietic and non-hematopoietic tissues (Table 2
) [7
]. Usually multiple members are expressed in the
same cell or tissue. They participate in a variety of cellular
processes: cytoskeletal assembly and organization, cell-cell contact,
cell-matrix adhesion, induction of DNA synthesis, cell survival, and
cellular proliferation [7
]. Better understood in terms
of their mechanisms of activation than their function, Src PTK is
regulated by posttranslational phosphorylation and dephosphorylation
[8
]. Src kinases are also regulated by
ubiquitin-mediated proteolysis [8
].
So critical to a wide range of cellular behavior, protein tyrosine
phosphorylation was first described in studies on Src, just 20 years
ago [9
]. Studies on Src led to the identification of
receptor-encoded tyrosine kinases, signaling domains in a wide range of
molecules, and signaling complexes in a variety of subcellular
compartments. More recent insights into the regulation of Src,
identification of more Src substrates, binding partners, and signaling
cascades, and therapeutic inhibition of PTKs prompt this review of
Src-related PTKs in leukocyte biology [for other recent reviews, see
refs. 10 11
12
13
]. In this review, we will highlight the roles played by
Src PTKs in development, adhesion, phagocytosis, and survival in both
lymphocytes and myeloid cells. In sum, Src PTKs play a general role in
initiation and propagation of signals generated by a variety of
different receptor types.
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SRC FAMILY MEMBERS
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Members of Src family kinases found in mammalian cells are Src,
Fyn, Yes, Fgr, Lyn, Hck, Lck, B cell lymphocyte kinase (Blk), and Yrk
[11
, 14
15
16
17
18
19
20
21
]. Src’s importance was first
recognized as the retroviral oncogene that transformed chicken
fibroblasts, causing sarcomas [22
]. Yet, of the other
Src PTKs, only c-Yes and c-Fgr have a retroviral oncogene form. Src,
Yes, Fyn, and Lyn are widely distributed throughout the organism,
whereas Lck, Fgr, Hck, and Blk are confined to lymphoid and myeloid
tissues [23
]. Several of the Src PTKs, Fyn, Lyn, and
Hck, occur in alternatively spliced isoforms [24
25
26
27
28
29
].
The physiological significance of these isoforms and the redundancy of
Src PTKs remain elusive. Although the Src PTKs are found mainly in
association with the cell membrane and with other signaling molecules,
they may be found in other cellular compartments [30
].
Src, Fgr, and Lyn may be nuclear [23
,
31
32
33
], Hck is cytoplasmic [34
], Lck is
on the plasma membrane and pericentriolar vesicles in T lymphocytes,
and Fyn is on centrosomes and microtubule bundles [35
].
The role of Src PTKs in specific subcellular association is suggested
by the compartmentalized availability of substrates and binding
partners. The functional significance of different Src PTKs in
different compartments is not well understood.
|
STRUCTURAL FEATURES OF SRC PTKs
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Src PTKs share structural motifs, which are essential for their
function (Fig. 2
). Some of these structural motifs may be found in a wide range of
signaling molecules, including other non-receptor PTK (comparison of
motifs may be found in Table 3
). At the amino terminus of the Src PTK, the Gly (position 2) and
Cys (position 3) residues provide an acceptor site for the addition of
myristate and/or palmitate [36
, 37
]. This
posttranslational modification promotes protein association with the
lipid membrane (all of the Src PTKs are found at the inner surface of
the cell membrane). For Src and Blk, no palmitoylation occurs, and it
is unclear what this absence means. Membrane insertion via fatty acid
acylation is required for transformation by v-Src [38
].
In addition to bringing Src PTKs physically close to receptors and
integral membrane proteins, this acylation permits them to localize to
a subdomain of the cell membrane, known as the lipid raft
[39
, 40
]. These lipid rafts have a high
cholesterol and glycolipid content and are enriched with signaling
molecules and glycosylphosphatidylinositol (GPI)-linked proteins
[41
]. GPI-linked proteins include CD14, CD16b, CD24,
CD48, CD52, CD55, CD58, CD59, CD66b, CD66c, CD67, CD73, CD87, CD157,
and Thy-1. Most of these proteins co-precipitate with Src PTKs, and
their cross-linking results in tyrosine phosphorylation events
[42
, 43
]. In sum, Src PTKs localize to and
contribute to membrane-cytosol areas of high signaling activity. The
least understood component of Src PTKs is their unique domain, the
amino-terminal 
60 amino acids that confer specificity. In Lck this
unique domain provides a motif to interact with CD4 or CD8
[44
]. In the same cell, Fyn associates with the T cell
receptor [45
].
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Figure 2. Structural and functional domains of Src. (A) Src consists of four
major domains: the unique region, the SH3 domain, the SH2 domain, and
the catalytic or SH1 domain. The unique region contains a conserved
motif for fatty acylation, which is uncommonly referred to as the SH4
domain. The carboxy-terminal portion of the SH1 domains has the
critical negative tyrosine phosphorylation site. The tyrosine residue
in the catalytic domain that serves as a positive regulatory site is
not shown. (B) Src PTK activity is negatively regulated by two
intra-molecular associations. The SH3 domain recognizes a poly-proline
helix motif at the SH2-SH1 juncture. The SH2 domain recognizes a
carboxy-terminal phosphotyrosine residue. A phosphatase, e.g., CD45,
hydrolyzes that phosphotyrosine, which results in change in physical
conformation and catalytic activity.
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The next key feature of Src PTKs is the presence of two domains, which
facilitate protein-protein interactions. Because these two domains were
first appreciated in Src PTK and then found in a wide variety of
signaling molecules, they have merited the labels SH for Src homology.
The SH3 domain consists of 40–70 amino acid residues and recognizes
proline-rich motifs (PRM) [46
47
48
]. These PRMs are
vaguely characterized as RXPXXP or PXXP, where R is arginine, P is
proline, and X is any amino acid. The solving of the SH3 domain by
crystallography and nuclear magnetic resonance spectroscopy reveals a
"clasped hands" interaction, whereby the PRM assumes a particular
helical appearance (poly-proline helix II) and interdigitates with
pockets and a salt bridge in the SH3 domain. Surprisingly, a
poly-proline helix II is found in Src itself at the beginning of the
catalytic domain. This intrinsic SH3-proline interaction is one of two
key mechanisms, which negatively regulates Src kinase activity.
Although the SH3-PRM interaction is weak, with an apparent binding
affinity in µM, this interaction may be important to capture
substrates for Src PTKs. It is interesting that the affinity of SH3
domains for HIV Nef in vitro does not predict kinase
activation by Nef in vivo [49
,
50
]. In coexpression experiments in Rat-2 fibroblasts,
association of Nef and Hck leads to kinase activation both in
vitro and in vivo. Coexpression of Nef with Lyn was
without effect, although Nef shows equivalent binding to full-length
Lyn and Hck.
The SH2 domain typically consists of 100 amino acids and binds with
high affinity to specific phosphotyrosine motifs [46
,
51
]. The SH2 domains and their cognate phosphotyrosine
motifs are found in a number of signaling molecules. Yet, only 0.01%
of total cell proteins are tyrosine phosphorylated, and not all of the
phosphotyrosine residues serve as a docking site for SH2-containing
proteins. The apparent affinity for an SH2 domain to its cognate
phosphotyrosine motif is in nM. Through an ingenious
method of affinity chromatography, Cantley and co-workers determined
the alphabet code, or "zip code" of SH2 recognition
[52
]. The three amino acids, carboxy-terminal to the
phosphotyrosine, determine the specificity. Src PTKs bind
phosphotyrosine in the context of YEEI, this is the optimal binding
sequence. Like Src’s intrinsic SH3-PRM interaction, there is an
intrinsic SH2-phosphotyrosine interaction that negatively regulates Src
kinase activity. It is this critical phosphotyrosine that is mutated in
v-Src and results in the constitutively active form of Src
[53
].
The SH1 domain constitutes the catalytic domain, which comprises the
carboxy-terminal 250 amino acids [54
]. Within the
catalytic domain are the positive autophosphorylation site (for Src,
Tyr 416) and the negative phosphorylation site (for Src, Tyr 527). The
Csk (carboxy-terminal Src kinase) and its related Chk (Csk homologous
kinase) phosphorylate that tyrosine site [55
,
56
]. It is interesting that no significant abnormalities
were found in Chk/Hyl mutant mice [57
], however,
Csk-deficient mice died during embryogenesis with severe neural tube
defects [58
, 59
]. Chk/Hyl did not affect
the activity of Src, Hck, and Fgr in cultured bone marrow cells
[57
]. The newly described membrane protein, Cbp (Csk
binding partner) recruits Csk to the lipid raft, and thus facilitates
the tyrosine phosphorylation of Src [60
]. How Cbp
becomes active is another mechanism, awaiting elucidation.
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SRC SUBSTRATES AND BINDING PARTNERS
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Many stimuli lead to the increased kinase activity of Src PTKs via
a variety of receptor types. These stimuli also lead to activation of
other PTKs, some of which may be downstream of Src (e.g., Btk or Syk)
and some may be independent of Src (e.g., Jak or receptor PTKs). The
substrate phosphorylation is guaranteed by activation of multiple PTKs
or by activation of specific PTK and its site-specific tyrosine
phosphorylation. Several biochemical features may predict whether a
protein may be Src substrate, with ultimate identification of a
bona fide Src substrate resting on the absence (near
absence) of phosphorylation in Src PTK-deficient cell line or tissue.
Substrates should be found in the subcellular locations where Src may
be found, e.g., proximate to the plasma membrane. Substrates may have a
proline-rich region, which serves as a docking site for the Src SH3
domain. Substrates may also be tyrosine phosphorylated and require
additional tyrosine phosphorylation mediated by Src. In that case,
substrate availability may be directed via Src SH2/phosphotyrosine
interaction. Although there does not appear to be a highly specific Src
SH3 PRM, there is one for Src SH2 domain. The preferred Src SH2 binding
motif is YEEI/L, where the Tyr is phosphorylated. In addition, the Src
substrate is characterized by a Tyr residue found within the highly
acidic milieu provided by repeated Asp or Glu residues. Src-like PTKs
may have a requirement for Ile or Leu in the position -1 with respect
to the phosphorylated tyrosine residue in position 0. Blk and Lyn have
a strong preference for a negatively charged amino acid in position +1,
but Src prefers Trp or Gly in this position [61
]. To
date, the main substrates of Src PTKs are adaptor molecules [Cbl, Crk,
p85 subunit of phosphatidylinositol 3-kinsase (PI 3-kinase), Shc,
Vav], cytoskeletal proteins (annexin II, ß- and 
-catenin,
paxillin, talin, vinculin, cortactin, AFAP110), enzymes [focal
adhesion kinase (FAK), Tec, phospholipase C (PLC), mitogen
activated protein (MAP) kinase, Ras-Gap], and nucleotide binding
proteins [signal transducer and transcriptional activator (STAT), Tcf,
Sam68] [11
].
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EXPERIMENTAL APPROACHES TO DECIPHER SRC PTKs PATHWAYS
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It has been a challenge to assign a specific function to a
particular Src PTK because of their redundancy in tissue expression and
commonality in signaling pathways activated by a variety of stimuli.
Much of what we believe is conjectural and contextual. One key approach
is to study the effects of gene deletion in either mutagenized cell or
in the whole organism (mouse knockouts).
The chicken DT40 B cell line has been widely appreciated; it is an
example of successful use of a cell line for somatic cell knockout
experiments [62
]. The DT40 cell line was derived from a
bursal lymphoma caused by infection from the avian leukosis virus and
subsequent deregulation of c-Myc [63
]. It is interesting
that this cell line undergoes homologous recombination at a high
frequency, so it has been used by a variety of investigators to study
the effects of somatic cell gene targeting. The DT40 cell line
expresses only one Src PTK (i.e., Lyn), making it highly relevant to
investigators of B cell receptor and Src signaling. We have modified
this cell line to analyze the Lyn PTK contribution to cytokine
[granulocyte colony-stimulating factor (G-CSF)] receptor signaling
[64
].
Most of what we believe to be specific features of Src PTKs has come
from mouse knockouts [23
]. Some of the results have been
surprising, even disappointing. Yes and Blk knockout mice are normal
[65
, 66
]. Hck-deficient or Fgr-deficient
mice have a few subtle myeloid cell deficiencies [67
,
68
]. The knockout of Src causes osteopetrosis and
impaired bone remodeling, which was unpredictable [69
].
Other Src PTKs gene targeting has resulted in mice with abnormal
lymphoid development (described below). A greater role for Src PTKs has
been appreciated when investigators created double or triple knockouts.
For instance, the Src/Yes and Src/Fyn double knockouts are lethal
[65
], the Hck/Fgr double knockout has compromised host
defense [68
, 70
], and Btk/Lyn double
knockouts have a more severe immunodeficiency than Btk knockout mice
[65
, 67
]. Two-thirds of Hck-/-
Src-/- double mutants die at birth; surviving animals
develop a severe form of osteopetrosis and show extreme levels of
splenic extramedullary hematopoiesis, anemia, and leukopenia
[71
]. These hematopoietic defects are caused by
abnormalities in the bone marrow environment because Hck-/-
Src-/- mutant stem cells reconstitute a normal
hematopoietic system in irradiated wild-type mice. Fgr-/-
Src-/- double mutants have no defects beyond those observed
in Src-/- animals. Fgr and Hck levels are increased in
Src-/- osteoclasts. Hck and Src serve partially overlapping
functions in osteoclasts, and the expression of Hck in Src-deficient
osteoclasts ameliorates their functional defects.
It is surprising to note that
Hck-/-Fgr-/-Lyn-/- mice are
moderately healthy and fertile. However, the total protein
phosphotyrosine level is greatly reduced in macrophages derived from
these mice. These cultured macrophages express normal levels of CD14
and no other Src-family kinases were detected. The analysis of both
elicited peritoneal macrophages (PEMs) and bone marrow-derived
macrophages (BMDMs) from triple-mutant mice shows lack of defects in
lipopolysaccharide (LPS)-induced activation. Nitrite production and
cytokine secretion [interleukin (IL)-1, IL-6, and tumor necrosis
factor 
(TNF-)] are normal or even enhanced in
Hck-/-Fgr-/-Lyn-/- macrophages
after LPS stimulation. The development of tumor cell cytotoxicity is
normal in triple-mutant BMDMs and only partially impaired in PEMs after
LPS stimulation. The activation of the ERK1/2 and JNK kinases, as well
as the transcription factor NF-
B, are the same in normal and mutant
macrophages after LPS stimulation [72
].
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SRC PTKs IN MEDIATING LEUKOCYTE DEVELOPMENT: T CELL RECEPTOR (TCR)
AS A PARADIGM
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The TCR consists of two subunits, most commonly the and ß
chains. TCR forms complexes with accessory molecules such as the
and 
chains and receptors such as CD3, CD4, or CD8
[73
]. Antigen activation of the TCR rapidly induces
changes in protein tyrosine phosphorylation of the immunoreceptor
tyrosine activation motif (ITAMS) found in the - and CD3-associated
chains (schematized in Fig. 3
) [45
, 74
, 75
]. The
initial events involve the activation of CD45 and its subsequent
hydrolysis of the carboxy-terminal phosphotyrosine of Fyn. Amplifying
the signal of the cross-linked TCR, Fyn then proceeds to phosphorylate
the ITAMS, which serve as docking sites for the tandem SH2 domains of
zeta-associated protein kinase-70 kDa (ZAP-70). Among the diverse
signaling events is the activation of PLC, which leads to hydrolysis
of phosphatidylinositol-4,5-bisphosphate and the production of
diacylglycerol and inositol trisphosphate. Diacylglycerol activates
protein kinase C (PKC), and inositol trisphosphate releases
intracellular calcium from the endoplasmic reticulum. Additional
molecules are recruited and different pathways activated, such as PI
3-kinase through Cbl, growth receptor binding partner 2 (Grb2)-Sos-Ras
through p36 adaptor molecule and/or Shc (Fig. 4
), and Jun amino-terminal kinase (JNK) through Vav-Rac. Cellular
responses include cell cycle progression and gene transcription.
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Figure 3. Activation of multimeric receptors. (A) For multimeric receptors, like
TCR, BCR, and the FcR, CD45 hydrolyzes the carboxy-terminal
phosphotyrosine of a Src PTK (1), e.g., Fyn, which in turn
phosphorylates the tandem Tyr residues comprising an ITAM (2). (B) The
phosphotyrosine residues serve as a docking site for the tandem
SH2-containing ZAP-70 or Syk (3). Binding of the ZAP-70 (or Syk) to the
ITAM results in its activation. (C) One of ZAP-70’s important
substrates is PLC, which then acts on
phosphatidylinositol-4,5-bisphosphate to generate diglyceride and
inositol trisphosphate (4), an example of cross-talk between tyrosine
phosphorylation and PKC/Ca2+ signaling networks.
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Figure 4. Src-Shc-Grb2-Ras-MAP kinase pathway. One important signaling pathway
controls proliferation. Src phosphorylates the adaptor molecule Shc,
which recruits Grb2 and the Ras exchange factor Sos. In turn, Ras
becomes activated, triggering the serine kinase cascade that includes
MAP kinase kinase, MAP kinase, and additional serine kinases, such as
p90Rsk. Src can also bypass Ras and activate Raf directly.
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Lck and Fyn have non-redundant roles in T cell function: whereas Lck
associates with CD4 and CD8 integral membrane proteins, Fyn associates
with TCR [45
]. Lck is expressed in pre-TCR thymocytes
and Fyn is expressed in mature thymocytes [76
]. Although
T cell leukemia has been described with constitutive activation of Lck,
neither Lck nor Fyn have been found defective in patients with T cell
immunodeficiency. The Lck knockout causes a T cell maturation defect
[77
78
79
]. The Fyn knockout causes defective TCR
signaling [80
, 81
]. Lck-deficient mice have
dramatic effect on T cell development, whereas Fyn-deficient mice have
a subtle defect.
Normal B cell development is regulated by other Src PTKs
[82
, 83
]. Blk (B lymphocyte kinase) is
expressed in B cell lineage and has a role in the B cell proliferation.
Transgenic mice expressing constitutively activated Blk (Y495F) show
malignant transformation in the B lymphoid progenitors characteristic
of proB/preB-I to preB-II transition [84
]. Expression of
constitutively active Blk in the T cells gave rise to clonal, thymic
lymphomas composed of intermediate single-positive cells. The
Lyn-deficient mice develop a lupus-like syndrome. They have reduced
number of peripheral B cells, lack the response to B cell receptor
(BCR) cross-linking or LPS activation, and with age mice develop high
serum IgM levels, lymphadenopathy, and splenomegaly [85
,
86
]. Playing a role comparable to Lck and Fyn in T cells,
Lyn promotes normal B cell development, including deletion of
autoreactive clones, and BCR function.
The role for Src PTKs in myeloid cell development is less clear, but
they do play a role in promoting growth factor-induced cell cycle
progression. Multiple studies show that Src PTKs amplify the
proliferative signals generated by RTK such as platelet-derived growth
factor receptor or the macrophage colony-stimulating factor receptor
[87
88
89
]. We have recently reported that the presence of
Lyn was required for G-CSF-induced proliferation in hematopoietic cells
[64
]. Subsequent studies identified Cbl and Shc as two
Lyn substrates, which were critical for proliferation
[90
]. Treatment with stem cell factor (SCF) leads to an
association of Lyn with the juxtamembrane region of its cognate
receptor, c-Kit, and an increase in Lyn activity [91
,
92
]. Application of Lyn antisense oligonucleotides or
PP1, an inhibitor, results in dramatic inhibition of SCF-induced
proliferation of hematopoietic cells. Src kinases may also contribute
to differentiation of myeloid cells. Expression of activated
hematopoietic cell kinase (Hck) blocked granulocytic differentiation of
32Dcl3 cells in response to G-CSF. These results suggest that
up-regulation of Hck expression is not required for granulocytic
differentiation [93
]. During urokinase-induced
differentiation of U937 cells, there is rapid and transient inhibition
of Hck and Fgr kinase activity and no change in Fyn or Lyn activity
[94
].
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SRC PTKs IN LEUKOCYTE ADHESION AND MIGRATION: INTEGRIN AS A
PARADIGM
|
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Neutrophils adhere to the vascular endothelium, migrate through
the vascular basement membrane, and accumulate at the involved site.
Monocytes/macrophage and lymphocytes also migrate and adhere
[95
, 96
]. Adhesion, migration, and
accumulation comprise a multi-step process mediated by the interaction
of a number of adhesion receptors on leukocytes and ligands expressed
on the other leukocytes and endothelial tissue: selectins,
gangliosides, integrins, and cell adhesion molecules [97
,
98
]. In terms of intracellular signaling, the
best-understood interaction is that mediated by the integrin receptors
expressed on the neutrophils and platelets [99
,
100
]. The functional integrin receptor is a dimer of
and ß subunits. There are 22 different integrin receptors made up
through combination of 17 different and 8 different ß subunits
[101
]. The very complex nature of integrin receptors has
been reviewed [100
, 102
]. Cross-linking of
integrin receptors in leukocytes induces adhesion, release of specific
granules and mediators, and up-regulation of proinflammatory cytokines.
Engagement of integrin receptors causes clustering and quickly triggers
changes in tyrosine phosphorylation of the and ß subunits
themselves and cytoskeletal-associated proteins (see Fig. 5
) [103
]. These include: FAK, paxillin, tensin, and
cortactin. Other molecules that regulate cytoskeletal changes are
phosphorylated: Cbl, Cas, and Vav. Integrin receptor complex recruits
FAK directly through its ß subunit and/or through its proximity to
paxillin and tensin. FAK undergoes autophosphorylation at Tyr397, which
then serves as a docking site for Src SH2. Src is then responsible for
amplifying FAK’s signal, and it is likely to be the PTK that
phosphorylates paxillin, tensin, Cbl, and Cas. One of the substrates
for Src is also FAK at Tyr925. This phosphotyrosine site serves as a
binding site for Grb2. FAK can itself associate with and activate PI
3-kinase, or Src can perform the same function via Cbl. Thus, integrin
signaling nicely describes the amplifying and diversifying effects of
Src that result in a network of events. Besides causing adhesion via
formation of focal contacts, this integrin-FAK-Src cascade induces
changes in anchorage independence, cell shape, cell cycle progression,
and gene transcription [104
105
106
].
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Figure 5. Src-Fak-paxillin complex. Some signaling cascades are extremely
complex. One important signaling complex regulates cellular adhesion.
Integrins activate Src that phosphorylates a tyrosine kinase Fak. In
turn, Fak phosphorylates a variety of cytoskeletal proteins, one of
which is paxillin. The formed protein complexes serve as a contact for
focal adhesions. Another signaling pathway controls cytoskeletal
rearrangement and migration. Src phosphorylates the Rac exchange factor
Vav, which activates Rac. In turn, Rac activates a serine kinase PAK
(for p21 activated kinase).
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|
Hck-deficient or Fgr-deficient cells have normal neutrophil response to
cross-linking of ß2 and ß3 integrins in
response to extracellular matrix proteins or monoclonal antibodies,
thus illustrating the redundancy of Src family members in the integrin
signaling pathway [67
, 68
]. In double
(Hck-/-Fgr-/-) and triple mouse knockouts
(Hck-/-Fgr-/-Lyn-/-), neutrophil
adhesion and migration is impaired [68
, 100
,
107
]. Lyn and Fgr in human neutrophils redistribute to
cytoskeletal fraction and colocalize with Syk during neutrophil
adhesion on fibrinogen-coated surface [108
,
109
]. The adhesion-dependent activation of neutrophils
can be affected by oxidants [110
]. Diphenylene iodonium,
an inhibitor of NADPH oxidase, or degradation of
H2O2 by exogenously added catalase inhibits the
adhesion-stimulated activities of Fgr and Lyn. When undifferentiated
promonocytic HL60 or U937 cells were stimulated with fibronectin or
anti-ß1 integrin antibody, Syk becomes activated and
tyrosine phosphorylated. In this process, Lyn becomes transiently
activated and co-localizes with the actin cytoskeleton
[111
, 112
]. Similarly, cross linking of
either CD34 or CD43, sialomucins coexpressed on hematopoietic
stem/progenitor cells, activates the signaling pathway for cytoadhesion
through Lyn and Syk [113
]. Stimulation of
undifferentiated hematopoietic KG1a cells with anti-CD34 or anti-CD43
induces homotypic cytoadhesion and formation of cap on CD34 and CD43,
respectively. The cap colocalized with F-actin. The tyrosine
phosphorylation of Lyn, Syk, pp60, pp69, and pp77 was increased at the
capping site [113
]. Similar findings were obtained in
monocytic U937 cells ectopically expressing CD34. In addition, normal
human CD34+ bone marrow cells showed cap formation of CD34
or CD43 after stimulation. Cbl, an adaptor molecule for Lyn plays an
important role in this process: after its phosphorylation, Cbl
associated with PI-3 kinase is translocated to the plasma membrane
fraction. Although Src kinases are clearly involved in cytoskeletal
organization, it is still not completely resolved if they are required
for extracellular matrix-mediated assembly of leukocyte adhesion
machinery (podosome) and cell motility or whether these kinases
transmit signals leading to these events [114
]. U937
with kinase-defective (K267M) Hck show enhanced adhesiveness and
F-actin redistribution. Hck is activated in macrophages after GPIs
stimulation [115
]. Hck is present on the azurophil
granules in human granulocytes and translocates toward the phagosomes
during phagocytosis. During
N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation
of human neutrophils or ATRA induction of NB4 cells only Lyn kinase is
activated. Zymosan or A23187 stimulation activates both Lyn and Fgr
[116
].
Leukocytes detect chemotactic gradients via GPCR, the best
characterized ones being receptors for C5a or fMLP. Although the
predominant biochemical effect of GPCR is activation of G proteins and
effector enzymes such as phospholipase A2 or adenyl
cyclase, cross-talking involving Src PTKs are well documented
[117
]. fMLP-mediated degranulation is markedly
diminished in neutrophils treated with the Src inhibitor PP1 or in Src
triple-deficient cell lines [118
]. Perhaps through the
ß subunits, Src PTKs couple GPCR to MAP kinase. An additional
pathway has been found to link GPCR to Src via the isoform of PI
3-kinase, which is activated by ß subunits [119
].
|
SRC PTKs IN PHAGOCYTOSIS: FCR AS A PARADIGM
|
|---|
Fc receptors for IgG (FcR), IgE (Fc
R), and IgA (FcR)
molecules are expressed on the leukocyte cell surfaces. Signaling
through these receptors defines host defense: phagocytosis, cell
cytotoxicity, and production and secretion of inflammatory mediators
[4
]. Neutrophils and macrophages are involved in
phagocytosis and macrophages and NK cells are involved in
antibody-dependent cell-mediated cytotoxicity. After the phagocyte has
migrated and accumulated at the site of infection, it engulfs the
opsonized bacteria, fungus, or virus. Phagocytosis proceeds through
activation of FcR. Three classes of these receptors and their
multiple isoforms have highly conserved extracellular domains and
distinct intracellular domains. Intracellular domains are of variable
length and they have one or two YXXL, conserved tyrosine regions. It is
interesting that FcRIIA that lacks a cytoplasmic domain, binds the
IgG-coated red blood cells, but it does not mediate erythrocyte
phagocytosis. Experiments in COS-1 cells transfected with different
isoforms and mutants of FcR showed that both the number and the
position of YXXL sequences within cytoplasmic domain are important for
phagocytosis. In addition, the differential distribution of FcR
isoforms correlate well with their function: isoforms that do not
mediate phagocytosis are expressed in lymphoid and myeloid cells that
are not phagocytic. Cross-linking of Fc receptors leads to
activation of tyrosine kinases [120
] and phosphorylation
of ITAMs within receptor cytoplasmic tails [4
].
Src, Fyn, Fgr, Lck, and Lyn are found in phagocytic cells, where they
are associated with the inactive FcRs [121
122
123
].
Src-deficient cells were less efficient than the wild-type cells in
mediating phagocytosis [124
]. Phagocytosis is dependent
on intact actin microfilaments [125
]. Cross-linking of
FcRI and FcRII on freshly isolated human monocytes leads to
transient phosphorylation of PTK Fgr, Syk and FAK, cytoskeletal protein
paxillin, and proto-oncogene Vav [126
]. It is
interesting that Hck-deficient and Fgr-deficient macrophages did not
have significant reduction in IgG-dependent phagocytosis. In contrast,
Hck-/Fgr-/Lyn-deficient macrophages had diminished or delayed
phagocytosis, respiratory burst, actin cup formation, and defective
activation of Syk and PI3-kinase. However, Hck, Fgr, and Lyn kinases
are not absolutely required for FcR-mediated phagocytosis because
the phagocytosis did occur, although at low level and delayed
[127
]. Instead, Syk is required for IgG-mediated
phagocytosis. Besides phosphorylating ITAMs and contributing to the
activation of Syk, recent evidence suggests another function of Src PTK
in phagocytosis that leads to changes in actin organization and
Ca2+ mobilization [both inositol trisphosphate
(IP3)-mediated and IP3-independent]. This is
confirmed by studies on Lyn’s role in the context of FcRI signaling
[128
129
130
]. Lyn is associated with the ß chain in the
inactive receptor and upon activation of receptor, transmits signals
for actin organization and Ca2+ mobilization
[131
]. In mast cells Lyn is involved in degranulation
mediated by FcRI where it associates with soluble tubulin
[132
].
|
SRC PTKs IN SURVIVAL: G-CSFR AS A PARADIGM
|
|---|
One of the major effects of the hematopoietins (such as G-CSF,
erythropoietin, or IL-3) is to maintain the survival by preventing the
apoptosis of blood cell progenitors [2
,
133
]. The factor-dependent 32Dcl3 myeloid cell line has
been used to study apoptosis. Retroviral expression of an activated
form of Hck markedly prolonged the viability of the factor-dependent
murine myeloid cell line, 32Dcl3, in the absence of IL-3 but failed to
abrogate the requirement for IL-3 for proliferation
[91
]. Expression of v-Src completely compensated for
IL-3-dependent survival and proliferation [134
].
Additional insights may be gleaned from children with severe congenital
neutropenia, who have a defective G-CSF receptor [135
,
136
]. The likely mechanism of this disorder and cyclic
neutropenia is defective anti-apoptosis and maturation
[137
, 138
]. Like that for the TCR,
integrins, or Fc receptor (FcR), engagement of the G-CSF receptor leads
to subunit dimerization and rapid changes in protein tyrosine
phosphorylation of itself and a variety of proximal signaling
molecules. The most likely candidates to mediate these phosphorylation
events are Janus and Src PTKs [139
]. Both families of
PTKs may be found in association with G-CSF receptor subunits. There
are four tyrosine residues in the carboxy-terminal domain of the G-CSF
receptor, and several of them become tyrosine phosphorylated. These
serve as docking sites for Grb2, STAT, and Lyn or Hck
[34
, 140
]. It is interesting that it is
this carboxy-terminal domain that is lacking in some patients with
severe congenital neutropenia [136
]. One major pathway
inhibiting apoptosis consists of
Src-Cbl-PI3-kinase-PI3-kinase-dependent kinase (PDK)-Akt-Bad
(Fig. 6
) and is activated in a variety of growth factor responses. In
Src-dependent fashion, activation of Akt is down-regulated by G-CSF
receptor’s carboxy-terminal region [141
].
View larger version (10K):
[in this window]
[in a new window]
|
Figure 6. Src-Cbl-PI 3-kinase pathway. One important PTK signaling pathway
inhibits apoptosis. Src phosphorylates the adaptor molecule Cbl, which
activates PI 3-kinase. In turn, a PI 3-kinase-dependent kinase
phosphorylates the serine kinase Akt, which phosphorylates Bad.
Serine-phosphorylated Bad uncouples from Bcl-2 and promotes resistance
to apoptosis.
|
|
Src PTKs may also contribute to the promotion of apoptosis. This
contradictory behavior is not that surprising because it is also
observed in the case of c-Myc [142
]. Studies in DT40
cells show that Lyn and Syk mediate signaling events involved in
radiation-induced apoptosis. Both Lyn and Syk are activated after
radiation exposure but with different kinetics: Syk being activated
first within 15 s and Lyn within 1 min [143
]. In
addition, DT40 cells deficient in Lyn or Syk undergo apoptosis,
suggesting that both kinases may not be required for radiation-induced
apoptosis. In contrast, IgM-induced apoptosis was blocked in DT40B
cells deficient in Syk or PLC2 but was still present in
Lyn-deficient cells [144
]. Adriamycin and etoposide,
inhibitors of topoisomerase II, caused apoptosis in DT40 wild-type
cells, Syk-deficient, and Btk-deficient cells but Lyn-deficient cells
become resistant. Expression of Fyn in Lyn-deficient cells restored
apoptosis. Lyn involvement in Topo II inhibitor-induced apoptosis is
independent of JNK [145
]. Lyn-deficient cells are also
resistant to ultraviolet-induced apoptosis [146
]. Like
Lyn, Lck also gets activated by radiation. This then leads to caspase-8
activation, which is independent of CD95 ligand expression in T
lymphoma cells [147
]. Ligation of both MHC-I and CD2 on
Lck-positive Jurkat cells leads to apoptosis [148
].
However, ligation of MHC-I on Lck-deficient Jurkat mutant cells
(JCaM1.6) did not have any influence on cell signaling or growth. Fyn
is also involved in apoptotic pathway in Jurkat cells. Lck mediates
TCR-induced apoptosis of mature cycling T cells by controlling Fas
ligand expression. Cells treated with Lck antisense oligonucleotides or
Lck-defective cell line was not able to induce apoptosis
[149
]. In response to Fas activation, CPP32 or
CPP32-like proteinase cleaves Fyn that translocates to the cytoplasm
[150
]. Activated T or B lymphocytes up-regulate the
expression of CDw150 receptor, which regulates apoptosis. SHIP and Fgr
associate with CDw150 cytoplasmic region. Activation of CDw150 induces
dephosphorylation of both SHIP and CDw150 and the association of Lyn
and Fgr with SHIP [151
]. Thus, Src PTKs either promote
or inhibit apoptosis depending on the type of stimulus or its context
[152
].
|
SRC PTKs AS DRUG TARGETS
|
|---|
As we have reviewed, Src PTKs are primarily involved in signal
initiation in hematopoietic cells. In addition, some of them have
tissue-restricted expression and therefore may be considered as
therapeutic targets in autoimmunity, allergic diseases, and cancer.
Signal transduction therapeutics based on interrupting tyrosine kinase
pathways has been proven through the clinical use of herceptin, a
monoclonal antibody that blocks ErbB2 activity in breast cancer. Signal
transduction inhibitor 571, a low-molecular-weight compound, inhibits
BCRAbl activity in chronic myeloid leukemia. Other pre-clinical studies
based on inhibiting Src PTKs by antisense oligonucleotides and
low-molecular-weight compounds have shown efficacy in cancer models.
For instance, to determine whether Lyn might play a role in supporting
acute myeloid leukemia growth, we analyzed fresh or cryopreserved
samples from patients. The majority demonstrated constitutive Lyn
activity. We have shown that targeting of Lyn by either the antisense
technique or Src-specific tyrosine kinase inhibitors will result in
profound inhibition of leukemic cell lines, such as HL60, TF-1, and
MO7e, or myeloid leukemic blasts [153
]. Small molecules
or antisense oligonucleotides directed against Lyn can also block
SCF-induced proliferation, granulocyte-macrophage colony-stimulating
factor-dependent neutrophil survival, or IL-5-dependent eosinophil
survival [154
155
156
]. Drugs used to treat cancer, e.g.,
methotrexate, cyclophosphamide, prednisone, and interferon, have found
applications in the management of autoimmune or chronic inflammatory
conditions. Based on these observations, inhibitors of Src PTKs and
their dependent pathways will be used in the near future for the
treatment of non-malignant leukocyte disorders.
|
ACKNOWLEDGEMENTS
|
|---|
Z. K. M. is supported by the Caligiuri Fellowship and
Hirtzel Foundation, and S. J. C. is supported by grants from
the National Institutes of Health, American Cancer Society, the
Leukemia and Lymphoma Society, the U.S. Department of Agriculture, and
the Pittsburgh Foundation.
Received July 22, 2000;
revised August 24, 2000;
accepted August 29, 2000.
|
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TOP
ABSTRACT
LEUKOCYTE SIGNAL TRANSDUCTION
