Bc319922089p

Vol. 274, No. 31, Issue of July 30, pp. 22089 –22094, 1999 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
Overactivation of Phospholipase C-␥1 Renders Platelet-derived
Growth Factor ␤-Receptor-expressing Cells Independent of the
Phosphatidylinositol 3-Kinase Pathway for Chemotaxis*
(Received for publication, January 13, 1999, and in revised form, May 6, 1999) ¨ nnstrand‡, Agneta Siegbahn§, Charlotte Rorsman, Matilda Johnell§, Klaus Hansen, and
Carl-Henrik Heldin
From the Ludwig Institute for Cancer Research, Biomedical Centre, Box 595, S-751 24 Uppsala, Sweden and the§Department of Clinical Chemistry, University Hospital, S-751 85 Uppsala, Sweden We have previously shown that porcine aortic endo-
sponsive cells leads to induction of mitogenicity, chemotaxis, thelial cells expressing the Y934F platelet-derived
and actin reorganization (for review, see Ref. 1). PDGF is a growth factor (PDGF) ␤-receptor mutant respond to
family of dimeric isoforms consisting of different combinations PDGF-BB in a chemotaxis assay at about 100-fold lower
of disulfide-bonded A- and B-chains. Thus, three isoforms of concentration than do wild-type PDGF ␤-receptor-ex-
PDGF exist with distinct binding characteristics toward the pressing cells (Hansen, K., Johnell, M., Siegbahn, A.,
structurally related PDGF ␣- and ␤-receptors. Binding of Rorsman, C., Engstro
¨ m, U., Wernstedt, C., Heldin, C.-H.,
PDGF to its receptors leads to dimerization of the receptors, an ¨ nnstrand, L. (1996) EMBO J. 15, 5299 –5313). Here
essential event in PDGF receptor activation (2, 3). Dimeriza- we show that the increased chemotaxis correlates with
tion leads to autophosphorylation on a number of tyrosine increased activation of phospholipase C-␥1 (PLC-␥1),
residues in the intracellular part of the receptors, providing measured as inositol-1,4,5-trisphosphate release. By
docking sites for a class of signal transduction molecules con- two-dimensional phosphopeptide mapping, the increase
taining Src homology 2 (SH2) domains, including members of in phosphorylation of PLC-␥1 was shown not to be se-
the Src family of tyrosine kinases, phosphatidylinositol 3-ki- lective for any site, rather a general increase in phos-
phorylation of PLC-␥1 was seen. Specific inhibitors of
nase (PI3-kinase), the GTPase activating protein of Ras (GAP), protein kinase C, bisindolylmaleimide (GF109203X), and
and phospholipase C-␥1 (PLC-␥1) (1).
phosphatidylinositol 3-kinase (PI3-kinase), LY294002,
In addition to undergoing autophosphorylation, the PDGF did not affect the activation of PLC-␥1. To assess
␤-receptor is also phosphorylated on one specific tyrosine resi- whether increased activation of PLC-␥1 is the cause of
due by members of the Src family of tyrosine kinases. Auto- the hyperchemotactic behavior of the Y934F mutant cell
phosphorylation of the PDGF ␤-receptor in the juxtamembrane line, we constructed cell lines expressing either wild-
region leads to association, phosphorylation, and activation of type or a catalytically compromised version of PLC-␥1
c-Src (4) and to a subsequent phosphorylation by Src of Tyr934 under a tetracycline-inducible promoter. Overexpres-
in the receptor (5). Mutation of Tyr934 to a phenylalanine res- sion and concomitant increased activation of wild-type
idue and expression of the mutant receptor in porcine aortic PLC-␥1 in response to PDGF-BB led to a hyperchemo-
endothelial (PAE) cells revealed an increased chemotactic re- tactic behavior of the cells, while the catalytically com-
sponse compared with cells expressing the wild-type PDGF promised PLC-␥1 mutant had no effect on PDGF-BB-
induced chemotaxis. Furthermore, in cells expressing
Blocking the association of the p85␣ subunit of PI3-kinase normal levels of PLC-␥1, chemotaxis was inhibited by
with the PDGF ␤-receptor, through mutation of Tyr740 and LY294002. In contrast, the increase in chemotactic re-
Tyr751 to phenylalanine residues, led to a significant reduction sponse seen upon overexpression of PLC-␥1 was not in-
of the chemotactic response in response to PDGF-BB (6), which hibited by the PI3-kinase inhibitor LY294002. These ob-
together with the observation that PDGF-induced chemotaxis servations suggest the existence of two different
is strongly inhibited by the PI3-kinase inhibitor LY294002 (5) pathways which mediate PDGF-induced chemotaxis; de-
suggests a role for PI3-kinase in mediating a chemotactic re- pending on the cellular context, the PI3-kinase pathway
or the PLC-␥1 pathway may dominate.
The present study was undertaken to investigate the mech- anisms behind the increased chemotactic response in the Platelet-derived growth factor (PDGF)1 stimulation of re- Y934F mutant cell line. We present data here that phosphoryl-ation and activation of PLC-␥1 are considerably increased inthe Y934F mutant receptor cell line. Furthermore, by the use of * This work was supported in part by a grant from the Nordic Cancer cell lines expressing either wild-type or a catalytically compro- Union, the Swedish Cancer Society, and the Axel and Margaret Ax:son mised mutant of PLC-␥1 under the tetracycline-inducible pro- Johnson Foundation. The costs of publication of this article were de- moter, we could show that the lipase activity of PLC- frayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 essential for the hyperchemotactic response and that cells over- U.S.C. Section 1734 solely to indicate this fact.
expressing PLC-␥1 are independent of PI3-kinase for PDGF- ‡ To whom correspondence should be addressed. Tel.: ϩ46 18 16 0406; Fax: ϩ46 18 16 04 20; E-mail: [email protected].
1 The abbreviations used are: PDGF, platelet-derived growth factor; DAG, diacylglycerol; HA, hemagglutinin epitope; IP , inositol-1,4,5- Materials—Precoated cellulose thin layer chromatography plates trisphosphate; PAE, porcine aortic endothelial; PH, pleckstrin homol-ogy; PI3-kinase, phosphatidylinositol 3-kinase; PLC- were purchased from Merck (Darmstadt, Germany), modified sequenc- C-␥1; SH2, Src homology 2; SH3, Src homology 3; PKC, protein kinase ing grade trypsin from Promega (Madison, WI). Polyvinylpyrrolidone was from Aldrich (Steinheim, Germany). Radionuclides were from Am- This paper is available on line at http://www.jbc.org
Phospholipase C-␥1-dependent Chemotaxis ersham Pharmacia Biotech (Buckinghamshire, UK). The Hunter thinlayer electrophoresis system was from C.B.S. Scientific Co. (Del Mar,CA). LY294002 was from Biomol (Plymouth Meeting, PA). Rabbit an-tiserum against PLC-␥1 was generated by immunizing rabbits with apeptide corresponding to the carboxyl terminus of bovine PLC-␥1 (7).
An antiserum against the hemagglutinin (HA) epitope tag was raisedby immunizing a rabbit with the synthetic peptide YPYDVPDYAGY-PYDVPDYA conjugated to keyhole limpet hemocyanin (KLH).
Construction of Cell Lines Containing PLC-␥1 under a Tetracycline- inducible Promoter—PAE cells expressing the PDGF ␤-receptor werestably transfected by electroporation with the regulatory plasmidpUHD 172–1-neo (8). G-418-resistant clones were screened by transienttransfection with the luciferase reporter plasmid pUHC 13–3 andtested for luciferase activity in the presence or absence of doxycycline.
Clones with low background expression of luciferase and high induc-ibility in the presence of doxycycline were chosen for transfection withthe reporter plasmid pUHD 10 –3 containing either wild-type PLC-␥1 ora lipase inactive PLC-␥1 (H335F/H380F), each containing a carboxyl-terminal HA-tag. Plasmids were co-transfected with pPKGpuro, pro-viding puromycin as selection marker. Puromycin-resistant clones werescreened by Western blotting for expression of PLC-␥1 in the presenceor absence of doxycycline, using either anti-PLC-␥1 antiserum or ananti-HA-tag antiserum. The clone used expressing wild-type PDGF␤-receptor and wild-type PLC-␥1 is thus denoted PAE/wt␤/PLC-␥1 , whereas the clone expressing wild-type PDGF-␤-receptor and lipasecompromised PLC-␥1 is denoted PAE/wt␤/PLC-␥1 Assay for Release of Inositol Phosphates—Six-well plates with semi- FIG. 1. Two-dimensional tryptic phosphopeptide maps of
PLC-␥1 from wild-type PDGF ␤-receptor-expressing PAE cells
or cells expressing the Y934F mutant PDGF ␤-receptor. Cells
were incubated in the presence or absence of 20 ng/ml doxycycline for were labeled with [32P]orthophosphate, stimulated with PDGF-BB or 24 h to induce protein expression, followed by overnight incubation with left unstimulated, lysed, and immunoprecipitated with an anti-PLC-␥1 2 ␮Ci of myo-[3H]inositol in 2 ml of inositol-free Ham’s F-12, containing antiserum. Immunoprecipitated protein was separated by SDS-gel elec- 0.3% fetal calf serum. In cases where bisindolylmaleimide or LY294002 trophoresis, electrotransferred to Hybond-C Extra, and digested in situ were used, inhibitors were added to the medium 60 min before stimu- with trypsin. Phosphopeptides were separated by electrophoresis at pH lation with PDGF-BB. Assay for PDGF-induced release of inositol phos- 1.9 in the first dimension and by chromatography using isobutyric acid phates was performed essentially according to Eriksson et al. (9).
chromatography buffer in the second dimension. A, PAE/wt␤ cells with- In vivo [32P]Orthophosphate Labeling of Cells—Cells were seeded at out PDGF-BB stimulation; B, PAE/wt␤ cells with PDGF-BB stimula- subconfluency 2 days before the experiment and starved for 24 h in 0.2% tion; C, PAE/Y934F cells without PDGF-BB stimulation; and D, PAE/ fetal calf serum. Cells were labeled with [32P]orthophosphate essen- Y934F cells with PDGF-BB stimulation.
tially as described by Hansen et al. (5).
In Situ Trypsin Digestion, Two-dimensional Phosphopeptide Map- ping, and Phosphoamino Acid Analysis—Phosphorylated proteins were phosphorylation sites appeared in cells expressing the Y934F separated by SDS-polyacrylamide gel electrophoresis, using an 8% gel mutant, cells expressing the wild-type PDGF ␤-receptor and and electrotransferred to nitrocellulose membrane (Hybond-C Extra, the Y934F mutant receptor were labeled with [32P]orthophos- Amersham Pharmacia Biotech). Samples were processed for tryptic phate. Following stimulation with PDGF-BB, cells were lysed and PLC-␥1 immunoprecipitated and processed for two-dimen- phoamino acid analysis, as described by Blume-Jensen et al. (10).
sional tryptic phosphopeptide mapping. A general increase in Cell Motility Assay—The chemotactic response of the different cell lines was assayed by means of the leading front technique in a modified the intensity of peptides phosphorylated in response to Boyden chamber, as described previously (11). In experiments where PDGF-BB could be seen in the Y934F mutant cell line, com- the effects of different inhibitors on the motility response were tested, pared with cells expressing the wild-type PDGF ␤-receptor the cells were preincubated for 10 min at 37 °C with the inhibitor at the (Fig. 1). Several peptides containing phosphotyrosine, as well indicated concentrations before the experiment. The inhibitors were as phosphoserine, appeared in response to PDGF-BB stimula- present throughout the experiment. In cases when doxycycline-induced tion (data not shown). No selective increase in the phosphoryl- protein expression was used, cells were incubated with 10 ng/ml doxy- ation of any peptide, nor any new phosphopeptides, were seen cycline for 24 h before the chemotaxis assay was performed. The che-motaxis assays were performed in Ham’s F-12, supplemented with 10% in PLC-␥1 from Y934F mutant cells.
Independence of PKC and PI3-Kinase for PLC-␥1 Activity— Mitogenicity Assay—PAE/wt␤/PLC-␥1 Previously, we have shown that the increased chemotactic re- cells were grown to confluency in 12-well plates and starved for 24 h in sponse seen in the Y934F mutant receptor cell line could be Ham’s F-12 containing 1 mg/ml bovine serum albumin, in the presence inhibited by bisindolylmaleimide, a protein kinase C inhibitor, or absence of 20 ng/ml doxycycline to induce protein expression. Me- while leaving the wild-type receptor-induced chemotactic re- dium was changed to the above medium containing 0.2 ␮g/ml [3H]thymi-dine and varying concentrations of PDGF-BB. Cells were incubated for sponse virtually unaffected. In contrast, an inhibitor of PI3- 24 h at 37 °C, after which cells were fixed with trichloroacetic acid and kinase, LY294002, effectively inhibited the chemotactic re- precipitated radioactivity was quantitated in a scintillation counter.
sponse seen in wild-type receptor-expressing cells, with littleeffect on the chemotactic response elicited by the Y934F mu- tant receptor (5). Therefore, we investigated the effect of bisin- Analysis of PDGF-BB-stimulated Phosphorylation of PLC- dolylmaleimide and LY294002 on PLC-␥1 activity in the two ␥1—We have previously shown that cells expressing a PDGF cell lines. Cells were labeled with [3H]myoinositol, incubated ␤-receptor with Tyr934 mutated to a phenylalanine residue, with the respective inhibitors, followed by stimulation with show enhanced chemotactic response compared with wild-type PDGF-BB and analysis of produced IP (Fig. 2). The inhibitor of receptor-expressing cells (5). It was also demonstrated that protein kinase C, bisindolylmaleimide, showed no effect on tyrosine phosphorylation of PLC-␥1 in response to PDGF-BB PLC-␥1 activity, neither in wild-type receptor cells nor in the was increased in cells expressing the Y934F mutant receptor.
To assess whether the increase in phosphorylation was selec- LY294002 slightly inhibited the PLC-␥1 activity in both cell tive for a particular tyrosine residue, and whether additional Phospholipase C-␥1-dependent Chemotaxis FIG. 2. Effect of LY294002 and GF109203X on PDGF-BB stimu-
lated inositol phosphate formation in wild-type PDGF ␤-recep-
tor-expressing cells and in cells expressing the Y934F mutant
FIG. 3. Effect of overexpression of wild-type PLC-␥1 and a
PDGF ␤-receptor. Cells were labeled with myo-[3H]inositol overnight
lipase-inactive mutant of PLC-␥1 on PDGF-BB-stimulated ino-
and stimulated with PDGF-BB. Released inositol phosphates were de- sitol trisphosphate release. Cells induced to express either PLC-␥1
termined. Cells were either treated with Me SO (control), 20 n construct by induction with 10 ng/ml doxycycline for 24 h, or non- GF109203X (GF), or 2.8 ␮M LY294002 (LY) induced control, were labeled overnight with myo-[3H]inositol in thepresence or absence of 10 ng/ml doxycycline. Cells were subsequentlystimulated Establishment of Stable Cell Lines Containing Wild-type or Dominant Negative PLC-␥1 under the Control of an InduciblePromoter—In order to investigate whether increased PLC-␥1activity causes increased chemotaxis and thus could explainthe hyperchemotactic phenotype of the Y934F mutant cell line,wild-type PDGF ␤-receptor-expressing cells were transfectedwith wild-type or catalytically compromised HA-tagged humanPLC-␥1 under the control of a tetracycline-inducible promoter.
Cell clones were analyzed for expression of PLC-␥1 in thepresence or absence of doxycycline. Conditions were optimizedso that the expression level of PLC-␥1 would lead to a response FIG. 4. Doxycycline induced expression of PLC-␥1 in the PAE/
in IP release after PDGF-BB stimulation similar to that seen wt␤/PLC-␥1
and PAE/wt␤/PLC-␥1
cell lines. Cells were left
in the Y934F cells (Fig. 3). The clones used expressed a negli- untreated or induced to express proteins with 10 ng/ml doxycycline for24 h. Cell lysates were separated by SDS-gel electrophoresis, followed by gible amount of HA-tagged protein in the absence of doxycy- electrotransfer to Immobilon-P filters. Filters were probed with either cline, while the protein expression was dramatically induced by anti-PLC-␥1 antiserum or anti-HA antiserum. IB, immunoblotting.
Overexpression of PLC-␥1 Leads to a Hyperchemotactic Re- sponse to PDGF-BB—Expression of PLC-␥1 was induced with Overexpression of Either Wild-type or Dominant Negative doxycycline for 24 h. Cells were then assayed for chemotaxis by PLC-␥1 Has No Effect on PDGF-BB-induced Mitogenicity in means of the leading front technique in a modified Boyden PDGF ␤-Receptor-expressing PAE Cells—In order to assess the chamber. In accordance with the Y934F mutant cells, PDGF possible role of PLC-␥1 in mediating the mitogenic response to ␤-receptor cells overexpressing wild-type PLC-␥1 responded to PDGF-BB, cells were incubated with doxycycline for 24 h to PDGF-BB with chemotaxis at concentrations almost 100-fold induce overexpression of either wild-type PLC-␥1 or catalyti- lower compared with control cells without induction of PLC-␥1 cally compromised PLC-␥1, followed by stimulation with vary- expression (Fig. 5A). In contrast, cells overexpressing a similar ing concentrations of PDGF-BB and incubation with [3H]thy- amount of a catalytically compromised version of PLC-␥1, in midine for 24 h. However, no effect was seen on PDGF-BB- which two histidine residues (His335 and His380) in the catalytic induced [3H]thymidine incorporation in cells expressing the domain had been mutated to phenylalanine residues, had ex- wild-type or the catalytically compromised PLC-␥1 (Fig. 7, A actly the same chemotactic response as control cells (Fig. 5B).
The Chemotactic Response in Cells Overexpressing PLC-␥1 Is Resistant to Inhibition by LY294002—Chemotaxis induced by PDGF-BB in wild-type PDGF ␤-receptor-expressing PAE cells We have previously shown that c-Src phosphorylates Tyr934 is efficiently inhibited by LY294002 (1). Similarly, chemotaxis in the second part of the kinase domain of the PDGF ␤-receptor was inhibited by LY294002 in the absence of doxycycline in (5). Cells transfected with a PDGF ␤-receptor mutant with cells expressing wild-type PLC-␥1 under a tetracycline-induc- Tyr934 replaced with a phenylalanine residue showed an in- ible promoter. In contrast, when cells were incubated with creased tyrosine phosphorylation of PLC-␥1 and an enhanced doxycycline to induce overexpression of PLC-␥1, the PI3-kinase chemotactic response to PDGF-BB. We have now further inhibitor LY294002 had no effect on PDGF-BB-induced chemo- shown that the enhanced chemotaxis coincides with an in- taxis (Fig. 6A). In contrast, when cells expressing a dominant crease in phosphorylation of PLC-␥1 on the same sites as those negative H335F/H380F mutant of PLC-␥1 under a tetracycline- phosphorylated in response to activation of the wild-type PDGF inducible promoter were incubated with doxycycline, the PI3- ␤-receptor. Kim et al. (12) have shown that phosphorylation of kinase inhibitor LY294002 effectively inhibited PDGF-BB-in- Tyr771 and Tyr783 in PLC-␥1 are essential for activation. No duced chemotaxis (Fig. 6B). Thus, the lipase activity of PLC-␥1 change in the stoichiometry in phosphorylation of Tyr1021, the is necessary to allow cells to migrate in the presence of PI3- primary association site for PLC-␥1 in the PDGF ␤-receptor, Phospholipase C-␥1-dependent Chemotaxis FIG. 5. Effect of overexpression of PLC-␥1 on chemotaxis. PAE/
doxycycline for 24 h. Chemotaxis toward PDGF-BB was assayed usingthe leading front technique in a modified Boyden chamber. A, PAE/wt␤cells with (●—●) or without (E—E) doxycycline; PAE/wt␤/PLC-␥1 FIG. 6. Effect of inhibition of PI3-kinase on chemotaxis in cells
cells with (f—f) or without (Ⅺ—Ⅺ) doxycycline; B, PAE/wt␤/PLC- overexpressing PLC-␥1. PAE/wt␤/PLC-␥1
cells with (●—●) or without (E—E) doxycycline.
cells (B) were incubated either in the presence (f—f) or absence (Ⅺ—Ⅺ) of 10 ng/ml doxycycline for 24 h. Cells were incubatedwith varying concentrations of LY294002 for 30 min at 37 °C prior to was seen in cells expressing the Y934F mutant compared with and during the chemotaxis assay. Chemotaxis was induced by 10 ng/mlPDGF-BB.
cells expressing wild-type PDGF ␤-receptor (data not shown).
The reason for the increased phosphorylation of PLC-␥1 in cellsexpressing the Y934F mutant PDGF ␤-receptor is not known; it ␤-receptor-expressing cells, inhibited by bisindolylmaleimide, is possible that the receptor kinase in the Y934F mutant has an inhibitor of protein kinase C, but resistant to inhibition by altered kinetics toward PLC-␥1 as a substrate. An additional the PI3-kinase inhibitor LY294002 (1). Here we demonstrate possibility is that the increase in phosphorylation of PLC-␥1, at that overexpression and enhanced activation of PLC-␥1 also least in part, could be due to the action of c-Src since it has been results in enhanced chemotactic response and that, under shown that c-Src is able to associate with and phosphorylate these conditions, the PI3-kinase inhibitor LY294002 has no PLC-␥1 (13, 14). We found that the increased phosphorylation effect on the chemotaxis induced by PDGF-BB. Taken together, of PLC-␥1 correlated with an increase in IP production (Fig.
these data suggest that at least two pathways can mediate 2). The magnitude of IP production produced in the Y934F PDGF ␤-receptor-induced chemotaxis, i.e. PI3-kinase and PLC- mutant cell line was about 3-fold higher than in wild-type ␥1. It is possible that the expression levels of PI3-kinase and receptor-expressing cells. The difference in response is not due PLC-␥1, and their magnitude of activation, determines which to differences in receptor number or ligand affinity between the cell lines, as indicated by Scatchard analyses of PDGF-BB Activation of PLC-␥1 leads to production of two second mes- sengers, IP and diacylglycerol (DAG). Binding of IP to recep- The importance of PLC-␥1 in chemotactic signaling was tors on the endoplasmic reticulum leads to release of calcium, pointed out by Kundra et al. (15, 16). However, PAE cells while DAG is an activator of the classical PKCs (18). Further- expressing a PDGF ␤-receptor mutant, with the association more, DAG has been implicated in stimulation of chemotaxis in site for PLC-␥1 Tyr1021 mutated to a phenylalanine residue, lymphocytes (19, 20). We could not detect any effect of the PKC showed unaffected chemotaxis toward the ligand (6). On the inhibitor bisindolylmaleimide on PLC-␥1 activity, neither in other hand, Wennstro¨m et al. (6) demonstrated that inhibition wild-type cells, nor in the mutant cell line (Fig. 2). This is in of PI3-kinase with wortmannin in PAE cells expressing PDGF contrast to findings by Ozawa et al. (21), who described feed- ␤-receptors led to inhibited chemotaxis. In contrast, Higaki et back inhibition of PLC-␥1 by PKC-␣ and PKC-⑀ in rat basophil al. (17) showed that, in vascular smooth muscle cells and Swiss RBL-2H3 cells. The PI3-kinase inhibitor, LY294002, had a 3T3 cells, inhibition of PI3-kinase had no effect on chemotaxis.
slight reducing effect on PLC-␥1 activity. This is consistent The chemotactic response seen in the Y934F mutant cell line with findings that full activation of PLC-␥1 requires binding of was, in contrast to the response seen in the wild-type PDGF phosphatidylinositol-3,4,5-trisphosphate to its pleckstrin ho- Phospholipase C-␥1-dependent Chemotaxis ␥1. Microinjection of DAG and IP did not stimulate DNA synthesis by themselves but did suppress the inhibitory effectof the SH2-SH2-SH3 polypeptide. These observations suggestthat both the SH2 and SH3 domains and the catalytic activityof PLC-␥1 are important for mediation of a mitogenic response.
In this report we did not observe any effect on PDGF-BB-induced DNA replication upon overexpression of either wild-type PLC-␥1 or a catalytically compromised version of thelipase.
In summary, we have shown that either PI3-kinase or PLC-␥1 are required for induction of chemotaxis by the PDGF ␤-receptor. Our findings suggest that it is the expression levelsof these individual enzymes and the extent of their activationthat determines which pathway that mediates the chemotacticresponse in individual cell lines. It is possible that the PLC-␥1and the PI3-kinase pathways are both needed for the chemo-tactic response, or one can substitute for the other given that itis expressed and activated at sufficient levels. Cross-talk be-tween the PLC-␥1 pathway and PI3-kinase pathway is knownon several levels. Full activation of PLC-␥1 was shown to re-quire the activity of PI3-kinase (22, 23, 28). Furthermore, Der-man et al. could show that products of PI3-kinase increased cellmotility through PKC (29). In addition to being activated byDAG, a product of PLC-␥1, PKC isoforms have been shown tobe phosphorylated and activated by PDK1, a downstream tar-get of PI3-kinase (30). Phosphorylation of the same conservedthreonine residue in the activation loop has previously beenshown to be essential to render PKC catalytically competent toautophosphorylate (31). Furthermore, PKC-⑀ has been shownto be activated by both PI3-kinase and PLC-␥1-dependentpathways (32). However, one cannot exclude the possibilitythat IP produced by PLC-␥1, leading to release of calcium from internal stores, plays a role in the increased chemotactic re- FIG. 7. Effect of PLC-␥1 overexpression on mitogenicity in-
sponse seen upon PLC-␥1 overexpression. An important future duced by PDGF-BB in wild-type PDGF ␤-receptor-expressing
goal will be to identify the common downstream signaling cells. Cells were incubated in the presence or absence of doxycycline for
molecules that mediate the chemotactic response seen after 24 h, followed by a 24-h incubation with [3H]thymidine in the presenceof varying concentrations of PDGF-BB, with or without doxycycline activation of PI3-kinase or PLC-␥1.
present. Labeled DNA was precipitated by trichloroacetic acid, and theincorporation of [3H]thymidine into DNA was measured in a scintilla- Acknowledgment—We thank Dr. Klaus Seedorf at the Hagedorn tion counter. A, PAE/wt␤/PLC-␥1 cells with (f—f) or without (Ⅺ—Ⅺ) Research Institute, Copenhagen, for cDNAs encoding wild-type PLC- doxycycline. B, PAE/wt␤/PLC-␥1 cells with (●—●) or without (E—E) doxycycline. FBS, fetal bovine serum.
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