Cell Architecture: from Structure to Function
Two functionally distinct pools of Src kinases for PDGF receptor signalling L. Veracini1 , M. Franco1 , A. Boureux, V. Simon, S. Roche and C. Benistant2 CRBM CNRS FRE 2593, 1919 Rte de Mende, 34293 Montpellier, France
Abstract The cytoplasmic tyrosine kinases of the Src family (SFK) play important roles in cell responses induced by growth factors, including cell growth, survival and migration. Here, we review how SFK participate in PDGF (platelet-derived growth factor) receptor signalling leading to DNA synthesis and actin assembly. Furthermore, evidence for a spatial compartmentalization of SFK signalling is also discussed.
SFK (Src family of protein tyrosine kinases) mitogenic signalling Several laboratories including ours have implicated SFK in PDGF (platelet-derived growth factor) receptor signalling for DNA synthesis, actin cytoskeleton rearrangement and receptor endocytosis [1]. How SFK regulate these biological responses is poorly understood. In the context of growth factor stimulation, SFK signalling is largely independent of the well known Ras/MAPK (mitogen-activated protein kinase) pathway. Rather, SFK phosphorylate specific substrates allowing c-myc expression for cell-cycle progression [1] (Figure 1). Several of these substrates have been identified including the adaptor Shc (Src-homology 2 domain containing transforming protein 1), the transcription factor Stat3 and the guanine nucleotide exchange factor Vav2 [1]. We have also reported that the cytoplasmic tyrosine kinase Abl is an important SFK mitogenic substrate [2]. This allowed us to propose a tyrosine kinase signalling cascade (PDGFR/ Src/Abl) for c-myc expression and DNA synthesis. Interestingly, this signalling pathway is dependent upon a functional p53, although the molecular mechanism responsible is currently unknown [2,3]. While Abl specific mitogenic substrates are not identified yet, we found that it operates on Rac/JNK (c-Jun N-terminal kinase) and Rac/Nox (NADPH oxidase) pathways for c-myc induction [4].
Figure 1 A model to show how SFK regulate PDGF-induced c-myc expression and DNA synthesis PDGF-induced SFK activation allows phosphorylation and activation of Abl, Shc, Stat3 and Vav2 for c-myc induction and DNA synthesis. Abl then operates on a Rac/JNK and Rac/Nox pathway for c-myc expression. The Ras signalling cascade may participate in mitogenesis through Myc protein stabilization and expression of other genes. MAPK, mitogenactivated protein kinase; MKK, MAPK kinase.
SFK morphological signalling In addition to mitogenesis, PDGF also induces F-actin circular dorsal ruffles (Figure 2A). This cellular process has been linked to macropinocytosis [5] and endocytosis [6] but a recent report pointed to an important function for cell migration into the extracellular matrix [7]. This cytoskeletal Key words: actin assembly, cell compartmentalization, DNA synthesis, growth factor, lipid raft, Src family tyrosine kinase. Abbreviations used: EDG, endothelial differentiation gene product; JNK, c-Jun N-terminal kinase; Nox, NADPH oxidase; PDGF, platelet-derived growth factor; S1P, sphingosine 1-phosphate; Shc, Src-homology 2 domain containing transforming protein 1; SFK, Src family of protein tyrosine kinases; WASP, Wiskott–Aldrich syndrome protein; WAVE, WASP family verprolin homology protein. 1 2
These authors have contributed equally to this work. To whom correspondence should be addressed (email
[email protected]).
rearrangement is specifically promoted by the activation of PDGFRβ (PDGF receptor β), although a similar effect could have been observed with EGF (epidermal growth factor). Dorsal ruffles are regulated by cortical actin polymerization through the activation of the Arp2/3 (actin-related protein 2/3) complex and involve the Rac effectors WAVE [Wasp (Wiskott–Aldrich syndrome protein) family verprolin homology protein] 1 and WAVE 2 [6]. While Rac plays a central role in actin assembly [7], additional pathways have been implicated including phosphoinositide 3-kinase, Ras C 2005
Biochemical Society
1313
1314
Biochemical Society Transactions (2005) Volume 33, part 6
Figure 2 A model to show how SFK regulate PDGF-induced mitogenesis and actin assembly (A) Left and middle: an example of F-actin staining of PDGF-stimulated NIH3T3 cells with rhodamine-phalloidin. Right: a three-dimensional reconstruction image of the actin ring obtained by image analysis (Imaris software). (B) PDGF activates a pool of SFK in lipid rafts/caveolae for c-myc induction and DNA synthesis and another pool outside lipid rafts/caveolae for actin assembly. Activation of this second pool is through cross-talk signalling: PDGF recruits S1K (sphingosine 1-kinase) to the membrane through a phospholipase Cγ (PLCγ )-dependent pathway for S1P formation. S1P further activates SFK outside lipid rafts/caveolae through an EDG receptor coupled to a heterotrimeric Gi protein allowing Abl activation for dorsal ruffle formation. Note that additional Src substrates (others) may be expected for actin assembly.
and Rab5 signalling [6,8]. Interestingly, we and others have recently identified SFK and tyrosine kinases of the Abl family as additional regulators of dorsal ruffle induction [2,9] (L. Veracini, M. Franco, A. Boureux, V. Simon, S. Roche and C. Benistant, unpublished work). These kinases may act, at least in part, by phosphorylating and regulating activity of actin binding proteins such as members of the WASP family, cortactin, dynamin and gelsolin [6]. C 2005
Biochemical Society
Compartmentalization of SFK signalling An important issue involves the mechanisms that govern SFK signalling specificity. This may be achieved by phosphorylation of specific substrates and/or recruitment of specific pools of SFK within the cell. In agreement with these hypotheses, PDGF-induced SFK activation was observed during recruitment to the plasma membrane with maximal activity at the cell periphery [10]. SFK are localized in distinct
Cell Architecture: from Structure to Function
subcellular compartments including perinuclear membranes, early endosomes, plasma membrane and cholesterol-enriched microdomains or ‘lipid rafts/caveolae’. These small organelles are composed of cholesterol, sphingolipids and proteins including caveolin, flotillin and glycosylphosphatidylinositol-linked proteins and signalling molecules including SFK and growth factor receptors [11]. While still a matter of debate, they are thought to act as lipid scaffolds for regulation of signal transduction [11]. By perturbing the lipid raft dynamic through membrane cholesterol depletion, we found two distinct pools of SFK involved in mitogenesis and actin assembly respectively (L. Veracini, M. Franco, A. Boureux, V. Simon, S. Roche and C. Benistant, unpublished work). Specifically, we found that SFK association with the PDGF receptor occurs in ‘lipid rafts/caveolae’ allowing catalytic activation, Stat3 phosphorylation, c-myc expression and DNA synthesis (Figure 2B). In contrast, PDGF-induced dorsal ruffles were not affected by membrane cholesterol depletion. Biochemical fractionation assays uncovered a separate pool of SFK outside ‘lipid rafts/caveolae’ that was linked to dorsal ruffle formation. In contrast with the SFK mitogenic signalling, PDGF stimulates this pool of SFK by a crosstalk mechanism implicating the lipid second messenger S1P (sphingosine 1-phosphate). Our observations support a model where PDGF induces S1P production through a phospholipase Cγ /sphingosine 1-kinase pathway. S1P then binds a seven transmembrane receptor of the EDG (endothelial differentiation gene product) family and stimulates SFK through the activation of a heterotrimeric Gi protein (Figure 2B) allowing actin assembly. A similar mechanism has been proposed for PDGF-induced lateral ruffle formation [12] suggesting that it may be a general mechanism for growth factors to induce cell migration. Additionally, our observations suggest that Abl is also an important substrate of SFK for dorsal ruffle formation [2] (L. Veracini, M. Franco, A. Boureux, V. Simon, S. Roche and C. Benistant, unpublished work). This raises the possibility that several pools
of Abl may be recruited for DNA synthesis and cytoskeleton rearrangement respectively. Accordingly, two distinct populations of active Abl have been visualized by fluorescence imaging in PDGF-stimulated cells [13]. Therefore, we believe that spatial compartmentalization of cytoplasmic tyrosine kinases is an important feature of cell signalling specificity. L.V. is supported by La Ligue Contre le Cancer, M.F. by Ministere ` de la Recherche and Association pour la Recherche sur le Cancer, A.B. by Association for International Cancer Research, and S.R. and C.B. by INSERM (Institut National de la Sante´ et de la Researche Medicale). ´
References 1 Bromann, P.A., Korkaya, H. and Courtneidge, S.A. (2004) Oncogene 23, 7957–7968 2 Furstoss, O., Dorey, K., Simon, V., Barilla, D., Superti-Furga, G. and Roche, S. (2002) EMBO J. 27, 514–524 3 Broome, M.A. and Courtneidge, S.A. (2000) Oncogene 19, 2867–2869 4 Boureux, A., Furstoss, O., Simon, V. and Roche, S. (2005) J. Cell Sci. 118, 3717–3726 5 Dharmawardhane, S., Schurmann, A., Sells, M.A., Chernoff, J., Schmid, S.L. and Bokoch, G.M. (2000) Mol. Biol. Cell 11, 3341–3352 6 Buccione, R., Orth, J.D. and McNiven, M.A. (2004) Nat. Rev. Mol. Cell Biol. 5, 647–657 7 Suetsugu, S., Yamazaki, D., Kurisu, S. and Takenawa, T. (2003) Dev. Cell 5, 595–609 8 Lanzetti, L., Palamidessi, A., Areces, L., Scita, G. and Di Fiore, P.P. (2004) Nature (London) 429, 309–314 9 Plattner, R., Kadlec, L., DeMali, K.A., Kazlauskas, A. and Pendergast, A.M. (1999) Genes Dev. 13, 2400–2411 10 Sandilands, E., Cans, C., Fincham, V.J., Brunton, V.G., Mellor, H., Prendergast, G.C., Norman, J.C., Superti-Furga, G. and Frame, M.C. (2004) Dev. Cell 7, 855–869 11 Simons, K. and Toomre, D. (2000) Nat. Rev. Mol. Cell Biol. 1, 31–39 12 Rosenfeldt, H.M., Hobson, J.P., Maceyka, M., Olivera, A., Nava, V.E., Milstien, S. and Spiegel, S. (2001) FASEB J. 15, 2649–2659 13 Ting, A.Y., Kain, K.H., Klemke, R.L. and Tsien, R.Y. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 15003–15008 Received 6 July 2005
C 2005
Biochemical Society
1315
