Bombesin, Vasopressin, and Endothelin Stimulation of Tyrosine Phosphorylation in Swiss 3T3 Cells IDENTIFICATION OF A NOVEL TYROSINE KINASE AS A MAJOR SUBSTRATE*

Neuropeptide-stimulated tyrosine phosphorylation of specific components in Swiss 3T3 cells was investi-gated using monoclonal antibodies directed against the src transformation-associated substrates p125 focal adhesion kinase (FAK), a novel type of cytosolic tyro- sine kinase, and p130. Treatment of Swiss 3T3 cells with the mitogenic peptides bombesin, vasopressin, and endothelin caused a striking increase in the tyro- sine phosphorylation of p125FAK, as judged either by anti-phosphotyrosine (anti-Tyr(P)) Western blots of a n t i - ~ l 2 5 ~ ~ ~ immunoprecipitates, or by a n t i - ~ l 2 5 ~ ~ ~ immunoblots of anti-Tyr(P) immunoprecipitates. Bom-besin-stimulated tyrosine phosphorylation of ~ 5 ~ was detectable within seconds and concentration-de-pendent (half-maximum effect of 0.3 nM). Neuropep- tides also stimulated the tyrosine phosphorylation of a second component of M, 130,000, previously identified as the major p130 phosphotyrosyl protein in src-trans-formed cells. Bombesin stimulated p130 tyrosine phos- phorylation with kinetics and concentration dependence similar to

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Non-receptor tyrosine kinases increase the phosphorylation of a distinct spectrum of cellular substrates compared with receptor tyrosine kinases (11)(12)(13). Tyrosine phosphorylation of multiple cellular proteins, including p125 and p130 components, is elevated in chicken embryo fibroblasts expressing activated variants of pp60"" and the oncogene products of other avian sarcoma viruses (11)(12)(13)(14). These transformationassociated substrates may play a role in the pathways that lead to transformation by pp60""" (13,14). Recently, Schaller et al. (14) identified p125 as a novel cytosolic tyrosine kinase, which is associated with focal adhesions and hence has been named Focal Adhesion Kinase (FAK). In contrast to most other non-receptor tyrosine kinases, ~1 2 5~~~ lacks the structural motifs that determine association with either membranes or other cellular proteins, including src homology-2 (SH2) and SH3 domains or a consensus acylation site (14). Like p125FAK, p130 is a major tyrosine-phosphorylatedprotein in src-transformed fibroblasts and forms stable complexes with activated forms of pp60""" (11,13). The normal cellular function(s) of these proteins remain poorly understood.
In the present paper, we have used specific mAb raised against ~1 2 5~~~ and p130 (12) to investigate the possibility that these proteins could serve as targets for neuropeptidestimulated tyrosine phosphorylation.

EXPERIMENTAL PROCEDURES
Swiss 3T3 fibroblasts were maintained and propagated for experimental purposes as described (15).
Labeling with f5S/Methionine-Cells were washed twice with DMEM without methionine and then incubated for 16 h in 1 ml of DMEM containing [35S]methionine (20 p~, 50 pCi/ml).
Immunoprecipitations-Quiescent cultures of cells (1 x IO6) were washed twice with DMEM, treated with peptide factors in 1 ml of this medium as indicated, and lysed at 4 "C in 1 ml of a solution containing 10 mM Tris/HCl, pH 7.6, 5 mM EDTA, 50 mM NaC1, 30 mM sodium pyrophosphate, 50 mM NaF, 100 p~ Na3V04, and 1% Triton X-100 (lysis buffer). Lysates were clarified by centrifugation at 15,000 X g for 10 min and precleared by incubation with albuminagarose for 1 h at 4 "C. After removal of albumin-agarose by brief (10 s) centrifugation, the supernatants were transferred to fresh tubes for immunoprecipitation. The src-transformation-associated proteins ~1 2 5~~~ and p130 were immunoprecipitated for 3 h at 4 "C with 1 pg/ ml mAb 2A7 and mAb 4F4 (12), respectively. Phosphotyrosyl proteins were immunoprecipitated with the anti-phosphotyrosine (anti-Tyr(P)) mAb, 1G2 (Oncogene Science, Inc.) coupled to agarose (4,5). The 2A7 and 4F4 mAb were precoupled to protein A-agarose and rabbit anti-mouse IgG prior to immunoprecipitation. Immunoprecipitates were washed three times with lysis buffer and further analyzed by Western blotting.
Western Blotting-Treatment of quiescent cultures of cells with factors, cell lysis, and immunoprecipitations were performed as described above. After SDS-PAGE, proteins were transferred to Immobilon membranes (4,5). Membranes were blocked using 5% nonfat dried milk in phosphate-buffered saline, pH 7.2, and incubated for 3-5 h with either the Py20 anti-Tyr(P) mAb or with mAb 2A7 as indicated in phosphate-buffered saline containing 0.05% Tween 20 and 1 pl/ml antibody. Immunoreactive bands were visualized using '251-labeled sheep anti-mouse IgG. In extracts of Swiss 3T3 cells, we found that mAb 4F4 was not suitable for Western blotting.

Tyrosine Phosphorylation and Neuropeptides
Materials-Bombesin, vasopressin, and albumin-agarose were obtained from Sigma. VIC was from Peninsula Laboratories Inc. 2A7 and 4F4 mAbs were the generous gift of Thomas Parsons and Randy Vines, University of Virginia. Agarose-linked anti-Tyr(P) mAb was purchased from Oncogene Science Inc. Py20 anti-Tyr(P) mAb was from ICN. '2sII-Labeled sheep anti-mouse immunoglobulin G (15 pCi/ pg) and [3sS]methionine (1000 Ci/mmol) were from Amersham, U.K.
All other reagents used were of the purest grade available.

RESULTS AND DISCUSSION
To investigate whether the recently identified tyrosine kinase ~1 2 5~~~ can serve as a substrate for neuropeptide-stimulated tyrosine phosphorylation, quiescent Swiss 3T3 cells were incubated with 10 nM bombesin, 20 nM vasopressin, or 10 nM VIC for 10 min, and lysates of the neuropeptide-treated cells were immunoprecipitated with mAb 2A7, which recognizes ~1 2 5~~~ (12,14). The immunoprecipitates were analyzed by Western blotting with a specific anti-Tyr(P) mAb. Fig. lA (left) shows that bombesin, vasopressin and VIC caused a striking increase in the tyrosine phosphorylation of a single anti-p125 immunoprecipitable band. This tyrosine-phosphorylated component exactly comigrated with the band obtained by immunoprecipitation of ~1 2 5~~~ from parallel cell  I  I  I  I  I  I  I  I  I  I   I  I  I  I  I  I  I  I  I  is indicated with an arrowhead. lysates followed by immunoblot analysis using mAb 2A7 instead of anti-Tyr(P).
To further substantiate that neuropeptides stimulate tyrosine phosphorylation of p125FAK, anti-Tyr(P) immunoprecipitates derived from lysates of neuropeptide-treated cells were immunoblotted with anti-p125 mAb. Similar to the results shown in Fig. L4, bombesin, vasopressin, and VIC caused a striking increase in the ~1 2 5~~~ immunoreactivity recovered by immunoprecipitation with anti-Tyr(P) (Fig. lA, right).
Tyrosine phosphorylation of ~1 2 5~~~ was an extremely rapid consequence of addition of bombesin to Swiss 3T3 cells. An increase in phosphorylation as revealed by anti-Tyr(P) immunoblotting of anti-p125 immunoprecipitates was detected as early as 30 s after addition of the peptide, reached a maximum within 1 min, and remained relatively constant for up to 1 h (Fig. 1B). Bombe~in-stimulatedp125~~~ phosphorylation was also concentration-dependent: half-maximum and maximum effects were obtained at 0.3 and 1 nM, respectively (Fig. IC). Immunoblotting with mAb 2A7 of anti-p125 immunoprecipitates prepared in parallel with those used for analysis of the kinetics and concentration dependence of tyrosine phosphorylation (Fig. 1, B and C, lower gel) verified that similar amounts of ~1 2 5~~~ were recovered after different times and concentrations of bombesin treatment.
To assess the approximate stoichiometry of bombesin-stimulated p125iFAK tyrosine phosphorylation, lysates prepared from control cells and from bombesin-treated cells were preincubated with anti-Tyr(P) mAb prior to immunoprecipitation of ~1 2 5~~~ with mAb 2A7. P125FAK was immunoprecipitated from parallel lysates after preincubating with rabbit antimouse IgG coupled to protein A-agarose instead of anti-Tyr(P) mAb. Recovery of ~1 2 5~~~ from depleted and undepleted lysates was monitored by Western blotting using mAb 2A7. As shown in Table I, the recovery of immunoreactive ~1 2 5~~~ from lysates of bombesin-treated cells was strikingly decreased from 87 k 11% to 22 f 6% (n = 4) by prior removal of tyrosine-phosphorylated ~1 2 5~~~. In contrast, preincubation of control lysates with anti-Tyr(P) mAb caused only a small decrease in the level of immunoreactive ~1 2 5~~~ (Table  I). These conclusions were further supported by experiments in which lysates were prepared from control and bombesin-

TABLE I
Effect of pre-incubation with anti-Tyr(P) mAb on the recovery of ~1 2 5~~~ from lysates of control and bombesin-treated cells Quiescent Swiss 3T3 cells were treated either in the absence (control) or presence of 10 nM bombesin for 10 min and lysed. The lysates were pre-incubated with anti-Tyr(P) mAb for 16 h, and subsequently the immunoprecipitates were collected and analyzed by Western blotting with mAb 2A7 (anti-Tyr(P) mAb). The supernatants of anti-Tyr(P) mAb immunoprecipitates were subsequently incubated with mAb 2A7 for another 6 h and then immunoblotted with mAb 2A7 (anti-Tyr(P) mAb + ~1 2 5~~~) .
Lysates from parallel cells were incubated with mAb 2A7 for 6 h after incubating for 16 h with rabbit anti-mouse IgG coupled to protein A-agarose instead of anti-Tyr(P) mAb and also analyzed by Western blotting with mAb 2A7. Autoradiograms of Western blots were scanned using an LKB Ultroscan XL densitometer. Values for ~1 2 5~~~ immunoreactivity are the means (-c S.E.) obtained from four independent experiments and are expressed as a percentage of the total immunoreactive ~1 2 5~~ recovered by immunoprecipitation with mAb 2A7. Similar amounts of ~1 2 5~~~ immunoreactivity were recovered by immunoprecipitation with mAb 2A7 from lysates of control and bombesin-treated cells.  (lanes 1, 3, Fi, and 7) or in the presence of 10 nM bomhesin (lanes 2,4,6, and 8 ) for 10 min and lysed. Some cell lysates were precleared of p130 by incubating for 3 h three times consecutively with mAb 4F4 using fresh antibody each time. These lysates were subsequently immunoprecipitated with anti-Tyr(P) mAb and analyzed by Western blotting with anti-Tyr(P) (lanes 5 and 6 ) . Lysates prepared from parallel cultures were immunoprecipitated either with ant Tyr(P) mAb (lanes I and 2), mAh 4F4 (lanes 3 and  4 ) , or mAb 2A7 (lanes 7 and 8), and the immunoprecipitates were then analyzed by Western blotting with anti-Tyr(P). The positions of p130 and p12sFAK and the phosphotyrosyl proteins, p90, p81, and p75 (4), are indicated by arrowheads. R and C, cells were incubated either with 10 nM homhesin for different times (panel R ) or for 10 min with different concentrations of bomhesin (panel C). The cells were then lysed, immunoprecipitated with mAh 4F4, and analyzed by Western blotting with anti-Tyr(P) as described. The position of p130 is indicated by an arrowhead. treated cells that had been labeled with ["S]methionine. Incubation of these lysates with mAb 2A7 resulted in the immunoprecipitation of a number of bands, including a major band which comigrated with ~1 2 5~"~. Similar to the results shown in Table I, recovery of ["S]methionine-labeled p12VAK from bombesin-treated but not control lysates was decreased by 70 f 3% ( n = 3) after preincubation with anti-Tyr(P) mAb. These results strongly suggest that bombesin stimulates tyrosine phosphorylation of ~1 2 5~"~ to a high stoichiometry.
T o establish that ~1 2 5~"~ is a constituent of the broad M , 110,000-130,000 phosphotyrosyl band stimulated by neuropeptides, ~1 2 5~"~ was removed from cell lysates prepared from bombesin-treated cells by preclearing with three consecutive incubations with mAb 2A7. Anti-Tyr(P) immunoblots of anti-Tyr(P) immunoprecipitates prepared from p125-immunodepleted lysates revealed a marked reduction in anti-Tyr(P) immunoreactivity of a band that exactly comigrated with ~1 2 5~"~. In contrast, immunodepletion of ~1 2 5~"~ did not affect bombesin-stimulated tyrosine phosphorylation of other components including a diffuse M , 130,000 band (results not shown), which comigated with a component present in anti-Tyr(P) Western blots of undepleted anti-Tyr(P) immu-noprecipitates ( Fig. 2 A , lanes 1 and 2). The relationship between the M , 130,000 band and a second src transformation-associated substrate, p130, was examined by immunoprecipitating lysates of bombesin-treated cells with a specific anti-pl30 monoclonal antibody, mAb 4F4, followed by immunoblotting with anti-Tyr(P). As shown in Fig. 2A  (lanes 3 and 4 ) , bombesin markedly increased the phosphotyrosine content of a major anti-pl30-immunoprecipitable band of M , 130,000. Furthermore, removal of p130 by incubating cell lysates with mAb 4F4, followed by anti-Tyr(P) immunoprecipitation and Western blotting, resulted in the depletion of a diffuse band which comigrated with the M, 130,000 phosphotyrosyl component present in anti-Tyr(P) and 4F4 immunoprecipitates (Fig. 2 A , lanes 3-6). In contrast, immunodepletion of pi30 did not reduce the level of a discrete M , 125,000 band (Fig. 2 A , lanes -5 and 6 ) , which comigrated with ~1 2 5 '~~ immunoprecipitated with mAb 2A7 (Fig. 2 A ,  lanes 7 and 8).
An increase in anti-phosphotyrosine immunoreactivity of p130 could be detected as early as 15 s after addition of bombesin, reaching a maximum after 1-2 min (Fig. 2R). Thereafter, p130 phosphorylation declined but remained above control unstimulated levels for up to 1 h. The effect of bombesin on p130 phosphorylation was concentration-dependent: half-maximal and maximum effects were obtained a t 0.5 and 1 nM, respectively (Fig. 2C). Vasopressin and VIC also markedly increased the tyrosine phosphorylation of p130 within seconds and a t nanomolar concentrations (results not shown). The concentration dependence of neuropeptide-stimulated p12sFAK and p130 tyrosine phosphorylation closely paralleled the concentration dependence for the effect of neuropeptides on the reinitiation of DNA synthesis (16)(17)(18).
Several reports have recently described neuropeptide stimulation of tyrosine phosphorylation in a variety of cell types, but neither the tyrosine kinase(s) nor the substrates involved were identified (4)(5)(6)(7)(8). The findings presented here demonstrate for the first time that the src transformation-associated substrates pl2sFAK and p130 are targets for neuropeptidestimulated tyrosine phosphorylation in Swiss 3T8 cells. The identification of p125'"K as a novel cytosolic tyrosine kinase with a unique structural architecture (14) suggests that this protein may function as a downstream element in a neuropeptide-stimulated tyrosine kinase pathway. Further experimental work will be necessary to elucidate whether p180 serves as a substrate for ~1 2 5~"~ and whether the increase in tyrosine phosphorylation of ~1 2 5~" " by neuropeptides is the result of autophosphorylation or the action of a more proximal kinase. Regardless of the precise mechanism(s), our results identify a previously unrecognized signal transduction link in the action of neuropeptide growth factors which act through G protein-coupled receptors.