(Received for publication, February 7, 1997, and in revised form, April 17, 1997)
From the Department of Physiology, Cornell University
Medical College, New York, New York 10021, the § Cell
Biology Program and Howard Hughes Medical Institute, Memorial
Sloan-Kettering Cancer Center, New York, New York 10021, the
¶ Department of Molecular Genetics, Kansai Medical University, 1 Fumizono-cho, Moriguchi 570, Japan, and the
McGill Cancer
Center, McGill University, Montreal, Quebec, Canada H3G 1Y6
The signal transduction pathway from heterotrimeric G proteins to the mitogen-activated protein kinase (MAPK) cascade is best understood in the yeast mating pheromone response, in which a serine/threonine protein kinase (STE20) serves as the critical linking component. Little is known in metazoans on how G proteins and the MAPK cascade are coupled. Here we provide genetic and biochemical evidence that a tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells. Targeted deletion of tyrosine kinase Csk in avian B lymphoma cells blocks the stimulation of MAPK by Gq-, but not Gi-, coupled receptors. In cells deficient in Bruton's tyrosine kinase (Btk), Gi-coupled receptors failed to activate MAPK, while Gq-coupled receptor-mediated stimulation is unaffected. Taken together with our previous data on tyrosine kinases Lyn and Syk, the Gq-coupled pathway requires tyrosine kinases Csk, Lyn, and Syk, while the Gi-coupled pathway requires tyrosine kinases Btk and Syk to feed into the MAPK cascade in these cells. The central role of Syk is further strengthened by data showing that Syk can bind to purified Lyn, Csk, or Btk.
Many neurotransmitters, hormones, chemokines, and sensory stimuli
initiate physiological effects through their membrane-bound seven-helix
transmembrane receptors that are coupled to heterotrimeric guanine
nucleotide-binding regulatory proteins (G proteins) (1-3). G proteins
transduce signals like an on and off switch. Liganded receptors
activate G proteins by catalyzing the exchange of GDP bound to the subunit (the inactive state) with GTP (the active state), resulting in
dissociation of
-GTP from the
subunits. Both
and
subunits can trigger downstream events (4). Several major intracellular
signaling pathways have been shown to be regulated by G protein-coupled
receptors. These pathways include the cAMP/protein kinase A pathway,
the phosphatidylinositol/calcium/protein kinase C pathway and the
mitogen-activated protein kinase (MAPK)1
pathway (1, 5, 6). The coupling mechanisms of G proteins to the cAMP
and phosphatidylinositol pathways have been defined (1). Gs
and Gi proteins directly modulate the activity of adenylyl cyclases, thus the cellular level of the second messenger cAMP. Gq and Gi proteins directly activate
phospholipase C
, thus increasing phosphatidylinositol turnover.
However, the mechanism by which G protein-coupled receptors activate
the MAPK pathway is less clearly defined in vertebrates and is an area
of intense research.
The MAPK pathway is found ubiquitously in eukaryotic organisms and is
used for regulating cell proliferation, differentiation, and many other
diverse biological functions (7-11). This cascade can be activated by
a variety of receptors, including G protein-coupled receptors (12-14).
The best understood coupling mechanisms to the MAPK pathway are from
receptors with intrinsic tyrosine kinase activity in invertebrates and
vertebrates (15-17) and from receptors coupled to heterotrimeric G
proteins in yeast (11). With receptor tyrosine kinases, extensive and
elegant biochemical and genetic work has led to the elucidation of the
coupling mechanism (15-17). Following ligand binding and receptor
autophosphorylation, the complex of the adapter protein Grb2 and the
guanine nucleotide exchange factor Sos, or the Shc (another adapter
protein)·Grb2·Sos complex, is recruited to
tyrosine-phosphorylated receptors. This leads to the prototype
Ras/Raf/MEK/MAPK pathway (7-10). In the yeast Saccharomyces
cerevisiae mating pheromone response, upon pheromone receptor
activation, an inactive heterotrimeric G protein dissociates, freeing
the subunits from an inhibitory
subunit.
is then able
to activate the MAPK cascade through a scaffold protein (STE5), a
serine/threonine protein kinase (STE20), and possibly other proteins
(11). Our understanding of the route leading from G protein-mediated
signals to the MAPK activation in vertebrate cells is much less
complete. A number of G protein-coupled receptors have been shown to
activate the MAPK cascade. Ras, Raf, protein kinase C,
Ca2+, and tyrosine kinase-dependent and
-independent pathways have been observed in certain cell types
(18-36).
We have begun a molecular genetic analysis of the activation pathway from G proteins to MAPK in vertebrates (30). We have chosen an avian lymphoma cell line DT40 as our model system due to the high efficiency of homologous recombination in these cells (37). Thus it is feasible for genetic manipulation in both generating loss-of-function mutants of relevant molecules by site-specific targeted deletion and in performing gain-of-function assay by overexpressing. Using tyrosine kinases Lyn and Syk knock-out cells, we have shown previously that the Gq-MAPK signaling requires Lyn and Syk, while the Gi-MAPK signaling needs Syk (30). Here with tyrosine kinases Csk and Btk knock-out cells, we demonstrate that while Csk is required for Gq-MAPK pathway, Btk is essential for Gi-MAPK signaling. Furthermore, with epistatic genetic and biochemical analysis in these tyrosine kinase-deficient cells, we have determined the order of action of these tyrosine kinases in the pathways. A surprising positive function for Csk, an otherwise negative regulator of Src-family tyrosine kinases, has been observed. Syk serves as a converging point and plays a central role in both Gq- and Gi-coupled signaling pathways. This point has been further strengthened by direct protein-protein binding analysis showing that Syk binds purified Lyn, Csk, or Btk. Therefore, these tyrosine kinases form a cascade that couples G proteins to the MAPK cascade in these avian lymphoma cells.
DT40 avian B lymphoma cells were grown in RPMI 1640 medium supplemented with 10% heat- inactivated fetal bovine serum, 1% chicken serum, 50 µM 2-mercaptoethanol, 2 mM glutamine, and 1% penicillin/streptomycin (30). Transient transfections were carried out with 2 µg of plasmid DNA and 5 µl of LipofectAMINE (Life Technologies, Inc.) reagent in six-well culture plates (30).
Immunoprecipitation and Immunoblot AnalysisDT40 whole cell extracts are prepared as follows: confluent cells are harvested from 10-cm plates, washed twice with cold phosphate-buffered saline, and pellets are resuspended in 0.8 ml of extraction buffer (150 mM NaCl, 10 mM Tris, pH 7.4, l mM EDTA, l mM EGTA, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 0.5% Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium orthovanadate, 0.02 mg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone, 0.03 mg/ml leupeptin). Resuspended pellets are passed five times through a 26-gauge needle, centrifuged at 5000 rpm for 5 min at 4 °C to remove insoluble material, and the supernatant is saved as the whole cell extract.
For immunoprecipitation, 10 µl of protein A-agarose (for polyclonal antibody) or protein G-agarose (for monoclonal antibody) was added to the whole cell lysate to preclear (38). Then 5 µl of primary antibody was added and continuously incubated at 4 °C for 30 min. After another 2-h incubation with 30 µl of protein A- or G-agarose beads, the immunocomplex was washed three times with the extraction buffer and three times with wash buffer (10 mM Tris, pH 7.4, 1 mM EDTA). The immunocomplex was then subjected to SDS-PAGE. Immunoblotting for ERK-1, Csk, Lyn, Syk, and Btk was done as described (30). Membrane filters were incubated in 1 × Tris-buffered saline, 5% milk for 1 h, then incubated in primary antibody for 2 h at room temperature. Blots were washed three times with Tris-buffered saline/Tween 20 and one time with Tris-buffered saline, then incubated with secondary antibody for 2 h at room temperature. Blots were washed again and signal was detected with ECL (NEN Life Science Products).
MAPK AssayExpression of mAChRs in transfected cells was
assessed by saturation ligand-binding analysis with intact cells using
the muscarinic antagonist [3H]methylscopolamine (39).
Untransfected DT40 cells showed virtually no detectable
[3H]methylscopolamine binding. m1 or m2 mAChR transfected
cells had ~40,000 binding sites/cell. After 24 h, transfected
cells were starved in serum-free medium for 12 h and whole cell
extracts were made after stimulation with carbachol (100 µM) for 5 min (30). ERK-1 immunoprecipitation was done
with a monoclonal antibody to ERK- 1 (Transduction Laboratory). The
MAPK assay was performed as described (30). 10 µg of MBP was used as
substrate. Kinase assay buffer was: 30 mM Tris-HCl, pH 8, 20 mM MgCl2, 2 mM
MnCl2, 10 µM ATP. The mixture was
preincubated 3 min before 10 µCi of [-32P]ATP was
added. After 10 min at 30 °C, samples were separated by 12%
SDS-PAGE. Gels were transferred to nitrocellulose membrane filters and
exposed for autoradiography. Western blot analysis was done with the
anti-ERK-1 monoclonal antibody. Quantitation was performed using a
Molecular Dynamics PhosphorImager.
Whole cell extracts were made after
stimulation with carbachol (100 µM for 20 s for m1
mAChR; 1 mM for 1 min for m2 mAChR). Csk
immunoprecipitation was done with a monoclonal antibody to Csk
(Transduction Laboratory), Lyn immunoprecipitation with a polyclonal
antibody to Lyn (40), and Syk immunoprecipitation with a polyclonal
antibody to Syk (Upstate Biotechnology, Inc.). Csk kinase assay was
done with 10 µg of -casein as substrate (41). Btk, Lyn, and Syk
kinase assays were done with 5 µg of the purified glutathione
S-transferase and the cytoplasmic domain of human
erythrocyte band 3 fusion protein (GST-CDB3) as substrate. The GST-CDB3
plasmid was constructed by polymerase chain reaction cloning of CDB3
from the plasmid pCDB3/T7-7 into pGEX-2T (42). The kinase buffer was:
50 mM HEPES, pH 7.4, 10 mM MnCl2, 1 mM phenylmethylsulfonyl fluoride, 10 µM ATP,
10 µCi of [
-32P]ATP. After 30 min at 30 °C,
samples were separated by 10% SDS-PAGE. Gels were transferred to
nitrocellulose membrane filters and exposed for autoradiography.
Quantitation was performed using a Molecular Dynamics
PhosphorImager.
Whole-cell extracts were dialyzed overnight at 4 °C in phosphate-buffered saline, 0.1% Triton X-100. For binding, 30 µl of nickel-nitrilotriacetic acid agarose beads with bound His-tagged protein (1~2 µg) was added to ~500 µg of whole-cell extract and incubated with gentle mixing for 1 h at 4 °C. The complex is pelleted by spinning at 10,000 rpm for 5 min and washed extensively with phosphate-buffered saline, 0.1% Triton X-100. The washed pellet was resuspended in 2 × sample buffer, boiled 5 min, spun down at 10,000 rpm for 2 min, and loaded onto a 12.5% SDS-PAGE gel. Separated proteins were transferred to nitrocellulose membrane for Western blot analysis.
To test whether the
COOH-terminal-Src kinase (Csk) plays a role in G protein signaling, we
have examined the vertebrate G protein-MAPK pathway in Csk-deficient
avian B lymphoma cells (DT40 cells) (43). MAPK was immunoprecipitated
with a monoclonal antibody against ERK-1, and its activity was measured
by an immune-complex kinase assay using MBP as substrate (30). Western
blot analysis with monoclonal antibodies to ERK-1 or ERK-2 revealed
that DT40 cells only express ERK-1, not ERK-2 (30). As we reported
previously, both Gq-coupled m1 mAChR and
Gi-coupled m2 mAChR can stimulate (~3-4-fold) the
activity of MAPK in DT40 cells after treatment with the agonist
carbachol (Fig. 1A). In Csk-deficient DT40 cells (Csk cells), the basal MAPK activity was similar to the
wild-type DT40 cells (Fig. 1, B and D).
Stimulation of m2 mAChR increased the MAPK activity in
Csk
cells to a similar level as it did in m2
mAChR-transfected wild-type DT40 cells (Fig.1B). However,
stimulation of m1 mAChR was unable to increase the MAPK activity above
basal level, despite similar amounts of mAChRs and MAPK in all these
cells (Fig.1B). To address whether the failure of
stimulation by m1 mAChR was the result of absence of Csk, we
transfected wild-type Csk back into Csk
cells. As shown
in Fig. 1B, Csk expression in
Csk
cells restored the stimulation of MAPK in response to
m1 mAChR. Epidermal growth factor receptor-mediated MAPK activation in
Csk
cells is not impaired (data not shown). These data
suggest that while Csk is required for Gq-coupled m1
mAChR-mediated activation of MAPK, it is not essential for
Gi-coupled m2 mAChR-mediated activation.
Btk Is Required for Gi, but Not Gq, Signaling to MAPK
We next examined the role of Bruton's tyrosine
kinase (Btk) in vertebrate G protein-MAPK pathway by studying the
activation of MAPK by Gq- and Gi-coupled
receptors in Btk-deficient cells (Btk cells) (44). The
Btk subfamily of nonreceptor tyrosine kinase contains one
pleckstrin-homology (PH) domain (45). Defects in Btk are responsible
for X chromosome-linked agammaglobulinemia in humans and X
chromosome-linked immunodeficiency in mice (for review, see Ref. 46).
The basal MAPK activity in Btk
cells was similar to the
wild-type DT40 cells (Fig. 1, C and D).
Stimulation of m1 mAChR increased the MAPK activity in
Btk
cells to a similar level as it did in m1
mAChR-transfected wild-type DT40 cells (Fig.1C). However,
stimulation of m2 mAChR was unable to increase the MAPK activity above
basal level (Fig. 1C). To address whether the failure of
stimulation by m2 mAChR was the result of absence of Btk, we
transfected wild-type Btk back into Btk
cells. As shown
in Fig. 1C, reconstitution of Btk restored the stimulation
of MAPK in response to m2 mAChR. Activation of MAPK by epidermal growth
factor receptor in Btk
cells is not affected (data not
shown). These data demonstrate that while Btk is required for
Gi-mediated activation of MAPK, it is not essential for
Gq-coupled activation.
Combined with our previous data showing the essential roles of Lyn and Syk in the Gq-MAPK pathway and of Syk in the Gi-MAPK pathway (30), it is clear that there are multiple tyrosine kinases involved in both Gq- and Gi-MAPK pathways. Therefore, we decided to determine the working order of Csk, Lyn, and Syk in the Gq-MAPK pathway and of Btk and Syk in the Gi-MAPK pathway.
Both Csk and Lyn Are Essential for Syk ActivationCsk was
originally purified as a kinase that negatively regulates the activity
of Src-family tyrosine kinases, including Lyn (47). Thus, it seems
puzzling that both Csk and Lyn are positively required for the
Gq-MAPK pathway. To address this paradox, we examined the
activation of Csk by Gq-coupled m1 mAChR in
Lyn cells and of Lyn in Csk
cells. Csk
kinase activity was measured by an immune-complex kinase assay using
-casein as substrate (41). Both m1 and m2 mAChRs can stimulate Csk
kinase activity in wild-type DT40 cells (Fig. 2,
A and E). In Lyn
cells, activation
of Csk by m1 mAChR is the same as in wild-type cells (Fig.
2B). In Csk
cells, Lyn kinase activity can
still be increased by m1 mAChR, despite a small increase of basal Lyn
activity in Csk
cells (43) (Fig. 2C). Thus the
activation of Csk and Lyn is independent of each other. Although the
small increase of basal Lyn activity indicates that Csk could
negatively regulate Lyn activity, the positive requirement of Csk for
the MAPK activation suggests that in Gq-MAPK signaling, Csk
acts mainly on a target(s) other than Lyn. A similar conclusion has
been recently reached from studies on normal lymphocyte
differentiation: both Csk- and Src-related kinases are positively
required, and Csk acts on targets other than Src-related kinases
(48).
We had previously shown that Lyn acts upstream of Syk in the
Gq-MAPK pathway (30). To determine the relationship between Csk and Syk in the Gq-MAPK pathway, we examined the
activation of Syk by m1 mAChR in Csk cells and of Csk in
Syk
cells (40). In Csk
cells, the
activation of Syk by m1 mAChR is greatly attenuated (Fig.
2D). In contrast, the activation of Csk in Syk
cells by m1 mAChR is the same as in wild-type cells (Fig.
2B). Thus Csk functions upstream of Syk. Therefore, both Csk
and Lyn are positively required for Syk activation by m1 mAChR, and
these two tyrosine kinases work in parallel.
Since both Btk and Syk are required for the
Gi-coupled m2 mAChR-MAPK pathway, we have determined the
relationship between Btk and Syk in the Gi-MAPK pathway.
Both m1 and m2 mAChRs can stimulate Btk kinase activity in wild-type
DT40 cells (Fig. 3, A and D). In
Syk cells, m2 mAChR is still able to increase Btk kinase
activity, although the basal and stimulated Btk activity is much lower
compared with wild-type DT40 cells (Fig. 3B). However, in
Btk
cells, m2 mAChR could not stimulate Syk kinase (Fig.
3C). Thus, it is likely that Btk functions upstream of Syk
in m2 mAChR-MAPK signaling. Since there are similar amounts of Btk
protein in wild-type and Syk
cells, our data also suggest
that Syk could serve as a positive feedback for maximal stimulation of
Btk by Gi-coupled receptors.
Functional Requirements for SH2, SH3, PH, and Kinase Domains
Both Csk and Btk have distinct structural domains such as
SH2, SH3, PH, and kinase domains. These domains play integral parts in
various aspects of signal transmission (45). Csk and
Btk
cells provide an excellent system to examine the
functional requirements of these domains in G protein-MAPK signaling.
Therefore, we did rescue experiments with domain-mutated tyrosine
kinases. Transfection of Csk
SH3 (SH3 deletion mutant), Csk
SH2
(SH2 deletion mutant), or catalytically inactive mutant
Csk(K
) constructs (49, 50) of Csk into Csk
cells did not restore the stimulation of MAPK by Gq-coupled
m1 mAChR (Fig. 4A). Addition of a
membrane-targeting signal, the myristoylation sequence from Src, to the
amino-terminal of Csk
SH3 (Src-Csk
SH3) (49, 50) did not
reconstitute the stimulation significantly. However, constitutive
membrane targeting of Csk
SH2 (Src-Csk
SH2) (49, 50) partially
rescued the response to m1 mAChR (Fig. 4A). These data
indicate that SH2, SH3 and kinase domains of Csk are essential for
coupling Gq to MAPK and that the function of the SH2
domain, at least in part, is to recruit Csk to the membrane, which is
consistent with the previous report (49, 50). We also tested Btk
mutants with dysfunctional PH, SH2, or kinase domains (44). None of
these domain mutants could rescue the response to
Gi-coupled m2 mAChR stimulation (Fig. 4B). These
results indicate that all three domains of Btk are essential for
Gi stimulation of MAPK.
Stimulation of Syk Kinase Activity by Overexpressing Lyn, Csk, and Btk
The above functional studies with loss-of-function mutants of
several tyrosine kinases indicate that these kinases act in a cascade
and that Syk sits at a converging point downstream of Lyn, Csk, and
Btk. To extend this observation further, we used a gain-of-function
assay and examined whether overexpression of Btk, Csk, or Lyn could
increase Syk kinase activity. As shown in Fig.
5A, overexpression of Lyn, Csk, or Btk led to
increased Syk kinase activity. Therefore, these data are consistent
with the genetic requirement of Lyn, Btk, and Csk for Syk
activation.
Syk Binds Purified Lyn, Csk, and Btk
Although the above genetic data pointed to the possibility of interactions of these tyrosine kinases, to test whether these tyrosine kinases interact directly, we have performed direct binding analysis with purified tyrosine kinases. To simplify the purification of the kinases, Lyn, Csk, and Btk cDNAs were subcloned into a vector with COOH-terminal hexahistidine tag. Purified Lyn-His6, Csk-His6, and Btk-His6 from Escherichia coli have protein kinase activities (detailed kinase characterization will be presented elsewhere).2 As shown in Fig. 5B, purified Csk, Lyn, and Btk bound to the endogenous Syk from the DT40 whole-cell extracts, while a control His6-tagged protein (NF-AT-His6, a T-cell-specific transcription factor) did not. We were also able to show binding of Lyn to Csk (data not shown). Btk did not bind to either Lyn or Csk (data not shown). These experiments demonstrate that Syk is able to form complexes with Lyn, Csk, and Btk. Thus these tyrosine kinases could form a multimolecular complex, as is the case with the MAPK module (11).
Our molecular genetic and biochemical analysis of the vertebrate G
protein-MAPK pathway places Syk kinase at a critical converging point
(Fig. 6A). Gq-coupled receptors
through Csk and Lyn, and Gi-coupled receptors through Btk,
activate Syk. Syk, in turn, feeds the signal into the MAPK cascade
(Fig. 6A). Although we have drawn the genetic pathway from G
protein to MAPK in DT40 cells as a linear pathway, it should be
stressed that the relationship among tyrosine kinases could be much
more complicated as exemplified by the feedback regulation of Btk by
Syk in the Gi-MAPK pathway.
The role of Btk and Csk in G protein-coupled receptor signaling is
different from their roles in B-cell receptor (BCR) signaling. In
Btk cells, although BCR-induced inositol
1,4,5-trisphosphate generation and intracellular calcium mobilization
were completely abolished, stimulation of MAPK by BCR was not affected
(44). Moreover, the overall pattern of tyrosine phosphorylation of
cellular proteins elicited by BCR was not different between
Btk
and wild-type DT40 cells (44). In Csk
cells, no defects in BCR signaling were observed, other than a small
increase in basal Lyn and Syk kinase activity (43). These data suggest
that these tyrosine kinases play different roles and participate in
different pathways in BCR and G protein-coupled receptor signaling.
Our surprising finding that Csk plays a positive role in the activation of MAPK by Gq signal and in Syk activation in DT40 cells is very intriguing. Csk was originally purified as a kinase that phosphorylates the COOH-terminal tyrosine residues of Src-family tyrosine kinases, including Lyn, thereby negatively regulating their activity (47). Overexpression of Csk decreased T-cell receptor-induced lymphokine production and protein tyrosine phosphorylation in T lymphocytes, although the in vivo function of Csk in T-cell receptor signaling is still not clear (49, 50). Mice deficient in Csk have increased activity of Src-family kinases and exhibit impaired formation of neural tube and embryonic lethality (51, 52). However, although Src-family kinases were activated, tumorigenesis was not observed in any tissues. Our data show that Csk is positively required for the Gq-MAPK pathway and that overexpressing Csk leads to increased, rather than decreased, Syk kinase activity. Furthermore, overexpressing Csk did not block the stimulation of MAPK by m1 or m2 mAChRs in wild-type DT40 cells.3 There are several recent reports suggesting that Csk has functions other than suppressing Src-family kinases. Overexpressing Csk in HeLa cells did not significantly suppress Src activity, but still led to dramatic changes in HeLa cell morphology (53). Csk is required for normal development of lymphoid cells. Csk deficiency blocks T- and B-cell differentiation as is the case with Src-family kinase deficiency. Thus Csk could act on targets other than Src-related kinases (48). It has also been shown that Csk could phosphorylate and activate the cell-surface receptor protein-tyrosine phosphatase (54). Therefore, Csk could perform dual positive and negative functions and work on Src-family kinases as well as other targets.
Could Syk Serve as a Scaffold Protein as Well as a Kinase?In
this report, we have presented data that demonstrate the central role
of Syk in both Gq and Gi signaling pathways to
MAPK. Overexpression of Lyn, Csk, or Btk leads to stimulation of Syk kinase activity. Purified Lyn, Csk, and Btk directly bind to Syk. Coimmunoprecipitation studies have indicated that additional cellular proteins can interact with Syk, including protein kinase Cµ (55, 56),
another Src-family member Lck (57), PLC1 and -
2 (44, 56), the
adapter protein SHC (58), as well as the B-cell receptor, the T-cell
receptor, and the high-affinity receptor for immunoglobulin E (46, 59).
It would appear from these various reports that the composition of
Syk-containing signaling complexes is dictated by the nature of the
activated receptor and the other signaling molecules involved. It is
possible that tyrosine phosphorylation of Syk, like tyrosine kinase
FAK, can create specific binding sites for SH2 domain-containing
proteins, thereby promoting the formation of intracellular multimeric
signaling complexes (60). Thus, Syk could function as a scaffold or
adapter protein as well as serve as a kinase. Recently it has been
shown that Syk-deficient mice exhibited perinatal lethality (61, 62).
It has been suggested that Syk has multiple functions in the mouse such
as in maintaining vascular integrity, in wound healing, and in B-cell
development (61, 62).
Our data demonstrate an essential role for nonreceptor tyrosine kinases in heterotrimeric G protein signaling. Although studies on tyrosine kinases have been very intense over the past decade, regulation of nonreceptor tyrosine kinases by heterotrimeric G proteins has not been studied until recently. Given the broad usage of both the heterotrimeric G protein signaling system and nonreceptor tyrosine kinases in cellular functions, it is very desirable to further our understanding how these two common signaling components communicate to each other. It is not surprising that tyrosine kinases play critical roles in many physiological responses mediated by G protein-coupled receptors. For example, smooth muscle contraction induced by mAChRs, angiotensin II, and vasopressin receptors is blocked by various tyrosine kinase inhibitors (for review, see Ref. 63). G protein-coupled angiotensin II receptor has been shown to stimulate tyrosine kinases Jak2 and Tyk2 in rat aortic smooth muscle cells leading to the activation of Jak/STAT pathway (64). A variety of G-protein-coupled receptors can stimulate the activity of tyrosine kinase FAK (65). Other documented G protein-coupled receptor-induced events that involve tyrosine kinases include chemotaxis through chemokine receptors, neuronal growth cone collapse by lysophosphatidic acid, cardiovascular hypertrophy/hyperplasia in hypertension, thrombin-induced platelet aggregation, focal adhesion assembly, and stress fiber formation (66-68).
Although the mechanism by which G proteins regulate the activity of
adenylyl cyclases and phospholipase C- is well understood, how G
proteins activate tyrosine kinases remains unknown. The activation
could be direct and indirect. Clearly, elucidation of detailed
activation mechanisms of these tyrosine kinases by G proteins requires
further biochemical studies. Regardless of the mechanism,
Gq- and Gi-mediated activations and interplays among the tyrosine kinases must be different, since both Gq
and Gi can stimulate all four tyrosine kinases, but their
pathways to MAPK are different. For example, both Gq- and
Gi-coupled receptors are able to stimulate Csk and Btk, but
the requirement of Csk and Btk for the Gq- and
Gi-MAPK pathways is different. We do not have a
satisfactory answer for how the specificity is achieved, as in the
cases of other complicated signal transduction pathways. Full
activation of many tyrosine kinases requires phosphorylation of
tyrosine residues (either through autophosphorylation or by another
tyrosine kinase) (69). It is likely that the tyrosine phosphorylation
event could serve as the activation mechanism. In the case of the first
tyrosine kinase in a tyrosine kinase cascade, other activation
mechanism(s) must exist. Since receptor tyrosine kinases and possibly
Raf kinase can be activated by oligomerization (70, 71), it is possible
that G proteins translocate tyrosine kinase to the membrane to increase
the local concentration leading to the activation of tyrosine kinase.
This notion is consistent with our results, indicating that membrane
localization of Csk is critical for its function in G protein
signaling. It is also possible that removal of the inhibitory mechanism
(either an inhibitory factor or a unfavorable conformation) can trigger
the activation of these kinases. Unraveling the molecular details of
the G protein-tyrosine kinase connection is one of the major challenges
for future studies.
Several very recent publications have provided strong evidence that, at least in some vertebrate cell types, activation of the MAPK pathway by G protein-coupled receptors is tyrosine kinase-dependent (30-34). The identity of the involved tyrosine kinases is beginning to be defined. We have used a genetic approach to reveal the roles of tyrosine kinases Lyn, Csk, Btk, and Syk in G protein-MAPK pathways in avian lymphoma cells. A dominant-negative mutant of another tyrosine kinase Pyk2 has been shown to interfere the signaling from Gq-coupled bradykinin receptor and Gi-coupled lysophosphatidic acid receptor to MAPK in PC12 cells (Ref. 34; but not in rat liver epithelial cell lines (72)). Src-family tyrosine kinases Fyn and Src have also recently been implicated in G protein-MAPK pathways in cardiac myocytes, smooth muscle, and COS cells (31-33). In avian lymphoma cells, we found multiple tyrosine kinases involved in G protein-MAPK pathways. In PC12 cells, Pyk2 is proposed to work together with Src to link G protein-coupled receptors to MAPK (34). The epidermal growth factor receptor, a receptor tyrosine kinase, has been implicated in transducing signals from G protein-coupled receptors to the MAPK cascade in Rat-1 cells (73). Another receptor tyrosine kinase, the platelet-derived growth factor receptor, has also been indicated to be involved in angiotensin II receptor-mediated mitogenic signaling in vascular smooth muscle cells (74). Therefore, different tyrosine kinases mediate the G protein signals in a general or cell type-specific way. However, the usage of tyrosine kinases in the G protein-MAPK pathway in some cells does not rule out a role for other tyrosine kinase-independent pathways in other cells. In fact, there is some evidence that in certain cells activation of the MAPK cascade by certain G protein-coupled receptors was not sensitive to tyrosine kinase inhibitors (21, 27, 36). Here we want to stress again the specificity of signaling pathways depends on the cellular environment, which is a very familiar theme to developmental biologists.
How do nonreceptor tyrosine kinases activate the MAPK cascade? At this point, we can only speculate that Syk, like receptor tyrosine kinases, can recruit adapter molecules such as SHC to transmit signals to the MAPK cascade (15-17). Several recent reports have suggested that tyrosine phosphorylation of SHC and the subsequently recruited Grb2·SOS complex are involved in activation of MAPK by Gq- and Gi-coupled receptors in a RAS-dependent pathway (28, 29, 75). Furthermore, Syk has been shown to associate with SHC (58). This is very similar to the mechanism proposed for FAK in linking the adapter protein Grb2 to RAS and MAPK activation in integrin signaling (60).
In comparison of the yeast and vertebrate G protein-MAPK pathways,
although both use protein kinases to link G proteins to the MAPK
pathway, yeast utilizes serine/threonine protein kinase STE20, while in
DT40 cells the linker is a tyrosine kinase cascade (Fig.
6B). Recent evidence suggests that the small GTP-binding protein CDC42 is required in the yeast pheromone response pathway (76,
77). Another small GTP-binding protein RAS has also been shown to be
required for the fission yeast Schizosaccharomyces pombe
pheromone response pathway (11) and for some G protein-coupled receptor-MAPK pathways in vertebrates (19-21). Future experiments with
knock-out DT40 cells will be necessary to determine any essential roles
for CDC42, RAS, G, and G
subunits in vertebrate
Gq- or Gi-coupled MAPK pathways.
We thank Dr. P. Low for the plasmid pCDB3/T7-7 and L. Chen for purified NF-AT-His6 protein. We are grateful to our colleagues and the members of our laboratory for reading the manuscript.