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J. Biol. Chem., Vol. 276, Issue 29, 26741-26744, July 20, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/29/26741    most recent C100229200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by He, H. Articles by Maruta, H. Search for Related Content PubMed PubMed Citation Articles by He, H. Articles by Maruta, H. Social Bookmarking                      What's this?

ACCELERATED PUBLICATION
Platelet-derived Growth Factor Requires Epidermal Growth Factor Receptor to Activate p21-activated Kinase Family Kinases^*

Hong He^§, Alexander Levitzki^¶, Hong-Jian Zhu, Francesca Walker, Antony Burgess, and Hiroshi Maruta

From the  Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Melbourne, Australia 3050 and the ^¶ Department of Biological Chemistry, Alexander Silvermann Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel

Received for publication, May 7, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The platelet-derived growth factor (PDGF) receptor (PDGFR) transactivates the epidermal growth factor (EGF) receptor (ErbB1) to stimulate the cell migration of fibroblasts through an unknown mechanism (Li, J., Kim, Y. N. & Bertics, P. (2000) J. Biol. Chem. 275, 2951-2958). In this paper we provide evidence that the transactivation of the EGF receptor (EGFR) by PDGFR is essential for PDGF to activate p21-activated kinase (PAK) family kinases. Fetal calf serum (10%) transiently stimulates the PAK activity in NIH 3T3 fibroblasts. The activation of PAK was completely inhibited by either PDGFR-specific inhibitor (AG1295) or EGFR-specific inhibitor (AG1478), suggesting that serum requires either the PDGF- or EGF-dependent pathway or the combination of both to activate PAK. PDGF-induced activation of PAK is completely inhibited by either AG1295 or AG1478, indicating that PDGF requires both PDGFR and EGFR for PAK activation. In support of this notion, a mouse embryo fibroblast cell line derived from the EGFR / mouse (from Dr. Erwin Wagner) doesn't activate PAK in response to PDGF. Expression of human EGFR in this cell line restores the ability of the PDGF to induce PAK activation. Our results indicate that PDGF activates PAK through transactivation of ErbB1.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Several distinct Ser/Thr kinases of the PAK¹ family such as PAK1, PAK2, and PAK3 are activated by CDC42/Rac GTPases and the SH3 proteins called PIX/Cool (1). PAKs are required for actomyosin-based membrane ruffling (2) and are essential for v-Ha-RAS to transform NIH 3T3 fibroblasts (3). We have shown previously that v-Ha-RAS requires both the EGF receptor (ErbB1) and ErbB2 to activate PAK through two independent autocrine pathways (3, 4).

In addition to the RAS-induced autocrine growth factors such as tumor growth factor-, HB-EGF, amphiregulin, and heregulin (3, 5-8), serum factors including PDGF are known to activate PAK (3, 9). Using a PDGF receptor (PDGFR) inhibitor AG1295 (10), an EGF receptor (EGFR; ErbB1) inhibitor AG1478 (10), and an ErbB2 inhibitor AG825 (10), as well as purified PDGF or EGF, we have characterized the biochemical nature of the serum-induced PAK activation in normal NIH 3T3 cells. Our data indicate that signaling from PDGF family ligands requires EGF receptor to activate PAK, through transactivation of EGF receptor (11-13).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells and Reagents-- The mouse E12.5 embryonic fibroblast cell line (EFE8-1) was derived from an ErbB1-deficient mouse (C57BL/6) generated by ErbB1 gene knock-out (14) and is a generous gift from Dr. Erwin Wagner (Research Institute of Molecular Pathology, Vienna, Austria). Both EFE8-1 cells and normal mouse NIH 3T3 fibroblasts (16) were grown in a standard medium, i.e. Dulbecco's modified Eagle's medium in the presence or absence of 10% fetal calf serum as described previously (3, 4, 15, 16). The PDGF receptor-specific inhibitor AG1295 and the ErbB1-specific inhibitor AG1478 were synthesized as described previously (10). The following antibodies were obtained from Santa Cruz Biotechnology, Santa Cruz, CA: anti-PAK antibody, anti-EGFR antibody, anti-PDGF- receptor antibodies, and anti-phospho-Tyr antibody. Anti-PDGF receptor antibodies were also purchased from PharMingen. Murine EGF was purified from mouse submaxillary glands in accordance with a published procedure (17). Purified human PDGF was purchased from Calbiochem.

Transfection of EGFR-deficient Fibroblast Cell Line EFE8-1 with Human EGFR-- To overexpress human EGFR in the EGFR-null mouse cell line EFE8-1, the cells were cotransfected with a pcDNA3 vector (Invitrogen) carrying M2-tagged human EGFR cDNA (18) and a second vector carrying puromycin-resistance selectable maker as described previously (16, 19). The FLAG tag was inserted after the leader sequence at residue 25 of EGFR. Clones expressing EGFR (EGFR/EFE8-1) were selected by puromysin resistance and immunoblotting with anti-M2 (18, 19) and anti-EGFR antibodies.

Assay for PAK Kinase Activity-- Serum-starved NIH 3T3, EFE8-1, or EGFR/EFE8-1 cells were stimulated by 10% serum, EGF (100 ng/ml), or PDGF (10 ng/ml) in the presence or absence of AG1478 (0.2 µM), AG825 (0.8 µM), or AG1295 (1 µM) for specified times. Cells were lysed in the lysis buffer (40 mM HEPES, pH 7.4, 1% Nonidet P-40, 1 mM EDTA, 100 mM NaCl, 25 mM NaF, 100 µM NaVO₃, 1 mM phenylmethylsulfonyl fluoride, and 100 units/ml aprotinin). The lysates containing 1 mg of protein (as measured by Bradford assay) were then immunoprecipitated with anti-PAK antibody (Santa Cruz), and the immunoprecipitates were subjected to the PAK kinase assay as described previously (2, 3). Immunoblotting was performed to determine the PAK protein level.

Immunoprecipitation and Immunoblotting-- Serum-starved EGFR/EFE8-1 cells were stimulated by either EGF (100 ng/ml) or PDGF (50 ng/ml) for 10 min in the presence or absence of either AG1478 (0.2 µM) or AG1295 (1 µM). Cell lysates containing 1.5 mg of protein (as measured by Bradford assay) were precleared with protein A-Sepharose beads (Amersham Pharmacia Biotech) and then incubated with either anti-EGF receptor (Santa Cruz) or anti-PDGFR antibodies (Santa Cruz) and protein A-Sepharose beads (13). The proteins in immunoprecipitates were separated on 7.5% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Micron Separations, Inc.). The membrane was blocked with 10% (w/v) skim milk in TBST (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween 20) at 4 °C overnight and then incubated for 1 h at room temperature with anti-phospho-Tyr antibody (Santa Cruz). After washing with TBST, the blot was incubated with horseradish peroxidase-conjugated anti-mouse secondary antibodies (Bio-Rad). The bound antibodies were visualized using ECL reagents (Amersham Pharmacia Biotech). To determine the amount of the receptors in the immunoprecipitates, the same membranes were stripped with 2% SDS and 100 mM dithiothreitol in 62.5 mM Tris, pH 6.4, for 30 min at 55 °C and washed with TBST. The blots were then reblotted with anti-EGFR or anti-PDGFR (PharMingen) antibodies.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Serum-induced PAK Activation Requires Both PDGF Receptor and ErbB1-- Within 10 min, fetal calf serum (10%) transiently activated PAK in serum-starved normal NIH 3T3 fibroblasts (Fig. 1A). Under these conditions, no increase in PAK protein levels is detectable. To identify the signaling pathway(s) involved in serum-induced PAK activation, we examined the effects of the following three specific tyrosine kinase inhibitors: the PDGF receptor inhibitor AG1295 (10), the EGF receptor inhibitor AG1478 (10), and the ErbB2-specific inhibitor AG825 (10). Either AG1295 (1 µM) or AG1478 (200 nM) inhibited completely the serum-induced PAK activation, whereas AG825 (800 nM) only partially inhibited the PAK activation (Fig. 1B). The particular concentrations of each inhibitor were chosen from published data (10), and our experiments demonstrated that the PAK kinase was not inhibited directly by any of these inhibitors (data not shown). These results indicated that serum-induced PAK activation required both PDGF receptor- and EGF receptor-dependent pathways. We resolved to determine whether serum-stimulated PAK activation involves PDGF receptor signaling transactivation of EGFR, which then requires ErbB2 in part to activate PAK, or EGF receptor signaling transactivation of PDGF receptor, which then requires ErbB2 in part to activate PAK.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   A, time course of serum-induced activation of PAK. Normal NIH 3T3 cells were cultured to confluence, serum-starved overnight, and then stimulated by 10% fetal calf serum for the indicated times. At each time point, the cells were collected and disrupted in lysis buffer. The cell lysates were subjected to PAK kinase assay as described under "Experimental Procedures." Similar levels of PAK protein were detected in each corresponding lane as judged by immunoblot (middle panel). The relative PAK kinase activity was calculated as -fold stimulation by serum over the control (non-stimulated) PAK kinase activity. The values in the graph are the means ± S.D. obtained from two independent experiments. B, the ErbB1, ErbB2, and PDGF receptor-specific inhibitors suppress serum-induced activation of PAK. Serum-starved NIH 3T3 cells were stimulated by 10% serum for 10 min in the presence or absence of AG825 (0.8 µM), AG1478 (0.2 µM), AG1295 (1 µM), and the combination of the three inhibitors, respectively. NS, no serum; S, 10% fetal calf serum. The values in the graph are the means ± S.D. obtained from three independent experiments.

PDGF Requires Both PDGF Receptor and ErbB1 for PAK Activation-- To clarify the above assumptions, we have examined the effects of PDGF and EGF on PAK activation separately. Like serum, PDGF (10 ng/ml) alone transiently activated PAK (Fig. 2A) as described previously (3, 9). The PDGF receptor inhibitor AG1295 (1 µM) blocked the PDGF-induced PAK activation (Fig. 2B). Surprisingly, the ErbB1-specific inhibitor AG1478 also completely inhibited the PDGF-induced PAK activation at the concentration of 200 nM. The results suggest that PDGF requires EGFR, in addition to PDGF receptor, for PAK activation. Although EGF (100 ng/ml) also transiently stimulates PAK activity (Fig. 3A), the EGF-induced PAK activation was inhibited by AG1478 (Fig. 3B). Clearly PDGF receptor activation is bypassed when EGF stimulates PAK activity. These observations strongly support our first hypothesis that PDGF receptor signaling transactivates EGFR. and they exclude the second hypothesis.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   EGF receptor is required for PDGF-induced PAK activation. Serum-starved NIH 3T3 cells were stimulated by PDGF for the indicated times (A) or for 10 min in the presence or absence of either AG1478 (0.2 µM) or AG1295 (1 µM) (B). The cell lysates were subjected to PAK kinase assay as described in Fig. 1. The relative PAK kinase activity was calculated as -fold stimulation by PDGF of the control PAK kinase activity. The values in the graph are the means ± S.D. obtained from three independent experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   EGF does not require PDGFR to activate PAK. Serum-starved NIH 3T3 cells were stimulated by EGF (100 ng/ml) for the indicated time (A) or for 10 min in the presence or absence of either AG1478 (0.2 µM) or AG1295 (1 µM) (B). The cells lysates were subjected to PAK kinase assay as described in Fig. 1. The relative PAK kinase activity was calculated as -fold stimulation by EGF of the control PAK kinase activity. The values in the graph are the means ± S.D. obtained from three independent experiments.

PDGF Does Not Activate PAK in Embryonic Fibroblasts from EGFR-deficient Mice-- Although the complete inhibition of PDGF-induced PAK activation by the EGFR-specific inhibitor AG1478 (200 nM) strongly supports the notion that PDGF requires EGFR for PAK activation in NIH 3T3 cells, these experiments clearly do not exclude the possibility that this compound at the concentration used inhibits the function of other cellular protein(s), which happen to be essential for both EGFR- and PDGFR-induced PAK activation. Therefore, to explore further the notion that PDGF requires ErbB1 to activate PAK kinase, we used the embryonic fibroblast cell line (EFE8-1) derived from ErbB1-deficient (/) mice (14). There was no PAK activation by PDGF in EFE8-1 cells although PDGF receptor is activated and becomes tyrosine-phosphorylated (Fig. 4A). Expression of human EGF receptor in this cell line restored the ability of PDGF to activate PAK (Fig. 4B). Again this response is inhibited by both AG1478 and AG1295 (Fig. 4B). Clearly the EGF receptor is required for PDGF-induced PAK activation.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   PDGF cannot activate PAK without EGF receptor. A, EGF receptor is essential for PDGF-induced PAK activation. Serum-starved EFE8-1 cells were stimulated by PDGF for the indicated time periods. No significant PAK activation was detected although the PDGFR was activated as shown by receptor tyrosine phosphorylation (top panel). The values in the graph are the means ± S.D. obtained from three independent experiments. B, EGF receptor expression restores PDGF-induced PAK activation. Serum-starved, EGFR/EFE8-1 cells were stimulated by PDGF (10 ng/ml) for 10 min in the presence or absence of either AG1478 (0.2 µM) or AG1295 (1 µM). PAK was transiently activated by PDGF in these cells. The activation of PAK was inhibited by both EGFR- and PDGFR-specific inhibitors. Similar levels of PAK protein were detected in each lane (middle panel). The values in the graph are the means ± S.D. obtained from three independent experiments. C, transactivation of EGFR by PDGFR. Serum-starved EGFR/EFE 8-1 cells were stimulated by either EGF (100 ng/ml; lane 5) or PDGF (50 ng/ml; lanes 2-4) for 10 min in the presence or absence of either AG1478 (0.2 µM; lane 3) or AG1295 (1 µM; lane 4). The cell lysates were immunoprecipitated (IP) with anti-EGFR antibody and then im-munoblotted (IB) with either anti-phosphotyrosine (PY) antibody (top panel) or anti-ErbB1 antibody (middle panel). Lane 1 is control (no added cytokine or drug). The Tyr phosphorylation of EGFR was clearly stimulated by PDGF, and the activation was inhibited by both EGFR- and PDGFR-specific inhibitors. The graph summarizes data from three independent experiments.

PDGF Receptor Transactivates the EGFR-- Although EGF activates PAK in NIH 3T3 fibroblasts (Fig. 3), the EGFR levels in this cell line are very low (20). Using the method described, we were unable to detect EGFR protein or EGF-mediated tyrosine phosphorylation of EGF receptor (data not shown). However, in the EGFR/EFE8-1 cell line, PDGF clearly stimulates tyrosine phosphorylation of the EGF receptor (Fig. 4C). The results indicate that the PDGFR is active in these cells following exposure to PDGF and that PDGFR transactivates EGFR, which is responsible for PAK activation. However, it still remains to be clarified how PDGF receptor activates EGFR.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the present study, we have observed that PDGF activates PAK via EGF receptor, and the EGF receptor becomes tyrosine-phosphorylated in response to PDGF treatment, suggesting that the transactivation of EGFR by PDGFR (11-13) is involved in PDGF-induced activation of PAK. PAK is required for normal cell motility and directed migration (21, 22). In particular PAK is required for PDGF-stimulated cell migration (23, 24). PDGF activates PAK, which then colocalizes with F-actin in lamelipodia and membrane ruffles, suggesting a role of PAK in directed cellular movement by regulating actin dynamics at the leading edge of cells. Recently it has been shown that PDGF-stimulated cell migration depends on the EGF receptor and that the EGF receptors become tyrosine-phosphorylated in respond to PDGF stimulation (13). This is consistent with our observations in the present study. Why does PDGF rely on the EGF receptor to induce cell migration? Our present observation suggests that the EGF receptor is required for PDGF to activate PAK, which regulates actin dynamics and consequentially leads to cell migration.

Like oncogenic RAS mutants, activation of the PDGF receptor directly activates PI3K (25), which in turn activates both CDC42 and Rac, which should lead to PAK activation (26-28). However, this well characterized PI3K pathway alone does not appear to be sufficient for PAK activation. We have demonstrated here that the PDGF receptor requires the EGF receptor for PAK activation. The oncogenic RAS mutants such as v-Ha-RAS also require the activation of the EGF receptor (3). Why do both PDGFR and oncogenic RAS require EGF receptor activation? Because both PDGFR and EGFR are able to recruit PAK to the plasma membranes through the SH2/SH3 protein NCK, which directly binds the N-terminal Pro-rich motif of PAK and the activated (Tyr-phosphorylated) PDGFR and EGFR (9, 25, 26), it is unlikely that PDGFR requires EGFR for the NCK-binding per se. Interestingly, the Ras-induced PAK activation absolutely requires a member of Src family kinases (3, 4). Src family kinases tyrosine-phosphorylate the CAT (Cool-associated tyrosine-phosphorylated protein) family proteins, which in turn bind to the SH3 family proteins called PIX/Cool (29). The SH3 family protein PIXs/Cools then bind with the N-terminal Pro-rich motif of PAK and stimulate PAK activity (30). Although both PDGFR and ErbB family kinases are known to activate Src family kinases (3, 4, 31), it still remains to be clarified whether the PDGFR-associated activation of PAK requires an ErbB kinase to activate Src family kinases.

Because the sensitivity of PDGF-induced PAK activation to the three specific tyrosine kinase inhibitors is basically identical to that of the serum-induced one, it is likely that the dominant PAK-activating factors in serum are PDGF family ligands but not EGF family ligands for the following reasons. First, 200 nM AG1478 does not inhibit either the PDGF receptor or ErbB2 as the IC₅₀ of AG1478 for both PDGF receptor and ErbB2 is higher than 100 µM (10). Second, as the IC₅₀ of AG1295 for both EGFR and ErbB2 is higher than 100 µM (10), 1 µM AG1295 does not inhibit ErbB family receptors. Finally, the IC₅₀ of AG825 for EGF receptor and PDGF receptor are 20 and 40 µM, respectively (10), again making it unlikely that 0.8 µM AG825 exerts any significant effect on these non-ErbB2 receptors.

	ACKNOWLEDGEMENT

We are very grateful to Dr. Erwin Wagner for a generous gift of the mouse ErbB1-deficient (/) fibroblast cell line EFE8-1 and Dr. Calle Heldin for critical reading of the manuscript.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Ludwig Inst. for Cancer Research, P. O. Box 2008, Royal Melbourne Hospital, Parkville, Melbourne, Australia 3050. Tel.: 613-9341-3155; Fax: 613-9341-3104; E-mail: Hong.He@ludwig.edu.au.

Published, JBC Papers in Press, May 16, 2001, DOI 10.1074/jbc.C100229200

	ABBREVIATIONS

The abbreviations used are: PAK, p21-activated kinase; EGF, epidermal growth factor; EGFR, EGF receptor; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PI3K, phosphatidylinositol 3-kinase; MBP, myelin basic protein.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 276/29/26741    most recent C100229200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by He, H. Articles by Maruta, H. Search for Related Content PubMed PubMed Citation Articles by He, H. Articles by Maruta, H. Social Bookmarking                      What's this?