(Received for publication, May 9, 1995)
From the
Receptors for chemoattractants that direct the migration of phagocytic leukocytes to sites of injury/infection also modulate many other leukocyte functions that are critical to the inflammatory response. These chemoattractant receptors, members of the G protein-coupled heptahelical receptor family, have been classically linked to cell activation via phospholipase C, calcium, and protein kinase C. We show here that activation of the N-formyl peptide chemoattractant receptor stimulates an additional protein kinase C-independent pathway through the Src-related tyrosine kinase, Lyn, in human neutrophils. We demonstrate that activation of Lyn is associated with binding to the Shc adapter protein, which becomes phosphorylated on tyrosine residues. This interaction appears to be mediated via the Shc SH2 domain. Complexes of phosphorylated Lyn and Shc with phosphatidylinositol 3-kinase are rapidly formed in stimulated neutrophils, correlating with phosphatidylinositol 1,4,5-trisphosphate formation and cell activation. This signaling pathway involving a Src-related kinase and the Shc adapter protein provides a potential mechanism linking chemoattractant receptors to downstream events involving Rac activation and NADPH oxidase. Regulation of Shc by G protein-coupled receptors may also allow these receptors to modulate the activity of the Ras/mitogen-activated protein kinase cascade.
The regulation of leukocyte function through the action of
chemoattractants and chemokines constitutes a critical aspect of the
body's immune response against microbial pathogens.
Chemoattractant receptors are members of the seven
transmembrane-spanning receptor family that utilizes heterotrimeric
GTP-binding proteins to transduce signals to the interior of the
cell(1) . The chemoattractant receptors of neutrophils,
including those for the N-formyl peptides (NFP), ()C5a, leukotriene B
, interleukin-8, etc., have
been shown to couple to ``classic'' signal transduction
pathways involving the activation of phospholipase C, formation of the
second messengers inositol 1,4,5-trisphosphate and diacylglycerol, and
the subsequent mobilization of Ca
and activation of
protein kinase C(2, 3) . It has only been recently
that evidence pointing to the existence of additional signaling
pathways has been obtained.
Phosphatidylinositol 3-kinase, which
generates the putative signaling molecule PIP, is activated
by chemoattractants(4, 5, 6) . PIP
formation is required for the stimulation of oxidant production
by the phagocyte respiratory burst (NADPH)
oxidase(7, 8) , and has been implicated in assembly of
the neutrophil actin cytoskeleton(9) . Neutrophils contain both
the p85/p110 PI 3-kinase, whose regulation by chemoattractant receptors
is not understood(10, 11) , as well as a novel G
protein
subunit-regulated PI 3-kinase whose contribution to
neutrophil activation has not yet been established(11) . The
mitogen-activated protein kinase cascade also becomes stimulated during
the course of acute neutrophil activation by chemotactic
agents(12, 13) . Levels of Ras GTP have been shown to
increase, and activity of the downstream kinases MEK and
mitogen-activated protein kinase (MAPK) is stimulated in response to
C5a and NFP(14, 15, 16) . The mechanisms
utilized by chemoattractant receptors to activate these pathways is
unknown.
Recently published reports have shown that a number of G protein-coupled receptors can activate the MAPK cascade through Ras-dependent mechanisms(14, 15, 16, 17, 18, 19) . Since these receptors are not known to couple to previously defined pathways for Ras activation, the means by which these receptors stimulate Ras function remain to be determined. In the present paper, we describe a novel pertussis toxin-sensitive, staurosporine-insensitive signaling mechanism used by chemoattractant receptors in which a nonreceptor tyrosine kinase, Lyn, and an SH2 domain-containing adapter protein, Shc, couple the N-formyl peptide receptor to PI 3-kinase and, potentially, to activation of the Ras/MAPK cascade and the respiratory burst oxidase.
Figure 3: Shc is present in Lyn precipitates from activated cells. Neutrophils were incubated for 1 min with 1 µM NFP (+) or control buffer(-) and then immunoprecipitated with anti-Lyn or anti-Shc antibody, as described under ``Materials and Methods.'' Precipitates were blotted for Shc as described. +/- in lane 1 indicates that an identical result was obtained +/- NFP. The position of Shc is indicated by the arrows. Results are representative of at least two similar experiments.
Figure 1:
Time course of phosphorylation of Lyn
and Shc, and of PI 3-kinase activity in Lyn, Shc, and Grb2
immunoprecipitates in NFP-stimulated neutrophils. PanelA, , Lyn autophosphorylation; an identical time
course was obtained using phosphorylation of enolase as the assay;
, Shc tyrosine phosphorylation; immunoprecipitates were blotted
for phosphotyrosine using the 4G10 monoclonal antibody (UBI) at 1:1000
dilution. PanelB,
,
,
, PI
3-kinase activity in Lyn, Shc, and Grb2 precipitates, respectively. The
initial activity at t = 0 min was set to 100% in each
panel. The results shown are representative of four
experiments.
Regulation of Lyn by the B
cell receptor and other cytokine receptors involves the interaction of
Lyn with a specific Src kinase binding motif present on these
receptors(31, 32, 33) . This so-called ARH1
motif is not present in the NFP receptor or other chemoattractant
receptors, suggesting that stimulation of Lyn does not result from
direct binding to receptor. Consistent with this, we could detect no
Lyn activity in NFP receptor precipitates from stimulated
neutrophils(34) . Stimulation of Lyn activity was dependent
upon activation of the G protein coupled to the NFP
receptor, as pertussis toxin effectively blocked Lyn activation (Fig. 2). In contrast, this response was not inhibitable by the
protein kinase C inhibitor staurosporine, indicating that this was not
a downstream effect resulting from protein kinase C activation by the
receptor (Fig. 2). Coupled with the fact that the Ca
ionophore A23187 did not stimulate Lyn activity (not shown),
these data indicate that Lyn activation is not a secondary response
resulting from activation of the classic chemoattractant signaling
mechanisms.
Figure 2: Effect of inhibitors on activation of the Lyn and Shc pathways. Cells were treated with pertussis toxin or staurosporine prior to stimulation with NFP as described under ``Materials and Methods.'' PanelA, effects on Lyn-stimulated phosphorylation of enolase; panelB, effects on tyrosine phosphorylation of Shc. Results shown are averages of two experiments.
We evaluated whether tyrosine-phosphorylated Shc interacted with other signaling proteins in NFP-stimulated neutrophils. The possible association of Lyn with Shc was probed by looking for the presence of Shc in Lyn immunoprecipitates and vice versa. The presence of Shc in anti-Lyn immunoprecipitates was observed by Western blotting (Fig. 3). A large percentage of total immunoprecipitatable Shc appeared to associate with Lyn in NFP-stimulated cells. In contrast, the presence of Shc could not be detected in Hck immunoprecipitates from activated cells (not shown). Consistent with the data of Fig. 3, we observed phosphorylated 53- and 56-kDa forms of Lyn when kinase assays were performed in Shc immunoprecipitates from NFP-activated neutrophils, but not in precipitates from unactivated cells (data not shown).
Figure 4: Lyn binds to the Shc-SH2 domain in stimulated neutrophils. Neutrophils were incubated for 1 min with 1 µM NFP or control buffer and then allowed to interact with a GST Shc-SH2 fusion protein as described, and the presence of Lyn was determined using Lyn kinase assay in the absence of exogenous enolase. Lane1, anti-Lyn antibody precipitate as a control to indicate the total amount of Lyn present; lanes2 and 3, unstimulated cells incubated with control GST-coated beads or Shc-SH2 beads, respectively; lanes4 and 5, NFP-stimulated cells with control or Shc-SH2 beads, respectively. Lane6 represents the NFP-stimulated cell lysate precleared with anti-Lyn antibody and then incubated with the Shc-SH2 beads. Results shown are representative of two experiments.
Figure 5:
Tyrosine-phosphorylated Shc associates
with other proteins in chemoattractant-stimulated neutrophils.
Neutrophils were incubated for 1 min with 1 µM NFP
(+) or control buffer(-) and then precipitated with anti-Shc
antibody, as described. Precipitates were blotted for phosphotyrosine
as described in Fig. 1. The positions of the unidentified
associated 25- and
145-kDa proteins are indicated by * and**,
respectively. Results are representative of three
experiments.
We demonstrate here a pertussis toxin-sensitive interaction
of the NFP chemoattractant receptor with a Src-related tyrosine kinase,
Lyn, and the Shc adapter protein during acute neutrophil stimulation.
Lyn, Shc, and PI 3-kinase rapidly associate in NFP-stimulated cells.
This association correlates with enhanced kinase activity of Lyn,
tyrosine phosphorylation of Shc, and elevation of PIP levels(5) . These events are not stimulated by elevations
in intracellular Ca
, nor blocked by staurosporine,
and they are thus not secondary to activation of protein kinase C. As a
further indication of the involvement of Lyn and Shc in acute
neutrophil signaling by chemoattractants, we also observed that the
levels of both Lyn and Shc increased (
3- and
3.5-fold,
respectively, by Western blotting) in HL60 cells differentiated with
Me
SO into neutrophil-like cells, providing circumstantial
support for their role in the signaling responses of mature cells.
The most likely signaling paradigm for the interactions we have just described is suggested by previous observations that Shc becomes active through tyrosine phosphorylation by c-Src(36) . We propose that the NFP receptor couples to Lyn, stimulating its autophosphorylation and kinase activity toward substrates. One of these substrates is the Shc adapter protein, which binds to phosphorylated Lyn via its SH2 domain and becomes tyrosine phosphorylated. Shc can then bind to and regulate PI 3-kinase activity. The basis for the interaction of Shc with PI 3-kinase remains to be defined, but it could involve the interaction of Shc directly with SH2 or SH3 domains on the p85 regulatory subunit of PI 3-kinase. When we evaluated whether PI 3-kinase bound to Shc-SH2 beads in stimulated neutrophils, we observed only slight increases in binding over controls, suggesting that Shc does not use this motif to bind to phosphotyrosines on the PI 3-kinase. This would be consistent with the observations of Vlahos et al.(10) , who found no stimulated PI 3-kinase activity in phosphotyrosine precipitates from activated neutrophils. Lyn has also been shown to interact directly with the PI 3-kinase p85 subunit through the binding of an SH3 domain on p85 to a proline-rich binding site on Lyn(33) . Alternatively, it is possible that the association of PI 3-kinase with Shc immunoprecipitates in stimulated cells is indirect, with Shc binding directly to Lyn, and Lyn then binding to the p85 subunit. This scenario has not been previously described and would place Shc upstream or parallel to Lyn in the transduction sequence from the NFP receptor, suggesting that Shc couples the receptor to the Lyn/PI 3-kinase pathway.
Based upon the time course and pertussis toxin sensitivity of the responses described herein, it is possible that regulation of PI 3-kinase activity through the Lyn/Shc interaction is involved with stimulation of downstream activities such as oxidant generation and chemotaxis. The requirement for PI 3-kinase activity in the signaling pathway leading to NADPH oxidase activation by chemoattractant receptors is indicated by studies with PI 3-kinase inhibitors(7, 8) . Accumulating evidence suggests that PI 3-kinase may be involved in activation of the Rac GTP-binding protein(40, 41, 42) . Rac is a critical regulator of oxidant production via the NADPH oxidase(26) . Furthermore, our results raise the probability that Shc adapter proteins play a role in linking chemoattractant receptors to the Ras pathway. The known ability of phosphorylated Shc to bind to Grb2 and the associated Ras GTP/GDP exchange protein, Sos, provides a potentially direct link between chemoattractant receptor signaling and the activation of Ras, which has been recently demonstrated in human neutrophils(14, 15) . The signaling paradigm we have described could also provide a mechanism to explain the ability of other G protein-coupled, seven transmembrane-spanning receptors to stimulate Ras activity. The endothelin and thyrotropin-releasing hormone receptors, also members of the heptahelical, G protein-coupled receptor family, have been recently reported to interact with Shc(43, 44) . Proof for the importance of the molecular interactions we have identified in intact neutrophil signaling will require the development of effective means to manipulate these cells at the molecular level; this capability is currently limited by the refractoriness of myeloid cells to conventional transfection methodologies.
In addition to possible
interactions with Grb2, the presence of an unidentified 145-kDa
tyrosine-phosphorylated protein was observed in Shc precipitates (Fig. 5). This protein may be the same as a 145-kDa protein
recently reported to bind to Shc in response to polypeptide growth
factors in hematopoietic cell lines (45) and in fibroblasts (46) . The function of this protein is not yet known, but it
interacts with a unique domain on Shc that binds phosphotyrosine and is
likely to be an additional mediator of chemoattractant signaling.
Activation of Lyn and Shc through the NFP receptor is pertussis
toxin-sensitive, indicating that the signaling cascade is initiated at
the level of the G protein(s) coupled to this and other
chemoattractant receptors. While the
subunits of G
have been shown to mediate the transduction of chemoattractant
receptor signals to phospholipase C
(47, 48) ,
little is known about the downstream effector targets of the
G
subunit in this system. It is tempting to speculate
that the G
subunit might couple to the Lyn/Shc pathway
to transduce additional downstream signals. Future studies directed at
the mechanisms by which G proteins couple to Lyn and Shc activation
should prove informative, as will further investigation of downstream
signaling elements linked to this pathway.