In addition to longer term effects such as cell proliferation and differentiation, stimulation of cultured fibroblasts with growth factors may result in rapid cytoskeletal reorganization. Stimulation of serum-starved fibroblasts with PDGF, EGF or insulin induces actin polymerization at the plasma membrane and subsequently stress fibre formation (155). EGF stimulation of the human carcinoma A-431 cell line induces microvilli within 30 seconds and membrane ruffles after 2-5 minutes (156). The most likely candidates for mediating the morphological effects of these growth factors are surface receptors with tyrosine kinase activity. The structure of protein tyrosine kinase receptors is typified by an extracellular domain (with either immunoglobulin-like, fibronectin type Ill-like, EGF-like, or cysteine-rich regions), a single transmembrane domain and an intracellular kinase domain (). Ligand binding induces receptor dimerization, followed by receptor 'autophosphorylation', mediated mainly by receptors within a dimer phosphorylating each other (157). Autophosphorylation sites within a non-catalytic domain (between the membrane and catalytic domain), the kinase domain (which in some cases increases the of the kinase activity) and the carboxyl- terminal domain serve as binding sites for proteins containing SH2 domains. SH2 domains bind short peptide motifs bearing phosphotyrosine residues, the specificity of the interaction being determined by residues surrounding the phosphotyrosine. SH2 domains can be divided into two groups depending the mechanism of their interaction with phosphotyrosine. One group binds to three residues immediately downstream of phosphotyrosines. The other group forms an extended hydrophobic groove that interacts with at least five amino acids.
The phosphorylation sites of the p-PDGF receptor have been studied extensively, with the protein containing at least ten phosphorylated tyrosine residues which interact with at least eight different signal transduction molecules. In this case protein binding to the
phosphotyrosine sites is highly specific. For instance, mutagenesis of Tyrio2i to Phe abolishes
PLCyi binding without affecting interactions with other SH2-containing proteins. However, this is not always the case as other phosphotyrosine sites bind multiple targets. Once bound to tyrosine kinase receptors, signalling molecules are activated by a number of mechanisms including tyrosine phosphorylation, as is the case with phospholipase Cy, induced conformational change, as with phosphatidylinositide 3-kinase (PI 3-K), or translocation to the plasma membrane, as with Ras (158).
Formation of receptor dimers allows diversity in the activation of different signalling pathways. PDGF exists as two forms (A and B) which bind to the PDGF-receptor as dimers. The PDGF-receptor also has two forms (a and P), with the a receptor containing different autophosphorylation sites compared to the p form, suggesting they activate different signalling pathways. AA PDGF dimers bind only a a PDGF receptors whilst BB PDGF binds a a and a p and p p PDGF receptors. Cells stimulated with AA PDGF and BB PDGF respond differently; BB dimer induces specific changes of the actin cytoskeleton and chemotaxis (157).
Many signalling molecules that contain SH2 domains also contain SH3 domains, including Src, PLC-Yi, Pf 3-K, p i20 RasGAP, Grb2, Crk. SH3 domains are also found in cytoskeletal proteins (including spectrin, myosin 1 and yeast-actin binding protein ABP-1) and components of the neutrophil oxidase p47phox and p67phox. The domain forms hydrophobic pockets which bind proline-rich peptides of approximately 10 amino acids. Binding specificity is determined by non-proline residues in the ligand which interact with variable loops in the SH3 domain (158). SH3 domains may be important for directing proteins to specific subcellular localizations as the SH3 domains of PLC-y and Grb2 micro-injected into
fibroblasts are targeted to microfilaments and membrane ruffles respectively (159).
A third binding motif, the pleckstrin homology (PH) domain, is also found in signaling proteins, including serine/threonine kinases, tyrosine kinases and their substrates, phospholipase C isoforms, regulatory proteins of small molecular weight GTPases, the GTPase dynamin and cytoskeletal proteins. The function of the domain is unclear but may target the proteins to membranes as some groups have reported phosphatidylinositol-4,5 Pg and other phospholipids binding to PH domains, whilst others have reported an association with the Py subunits of heterotrimeric G proteins (158).
1.15.2 Non-receptor Tyrosine Kinases.
A number of tyrosine kinase proteins are associated with the cytoskeleton. c-Src is localized to focal contacts of fibroblasts and the along with the related kinase, Fyn, is enriched in the growth cones of developing vertebrate neurons. Over-expression of a temperature sensitive allele of v-Src (a constitutively active form isolated from the Rous sarcoma virus) in chick embryo fibroblast induces reorganization of the cytoskeleton upon shifting to the permissive temperature, which include membrane ruffling, disruption of focal contacts and cell rounding and increased phosphorylation of ppl25^^, the fibronectin integrin receptor and paxillin (160). In vitro phosphorylation of the fibronectin receptor reduces the affinity of the receptor for the cytoplasmic protein talin and the extracellular protein fibronectin, a possible mechanism for the morphological changes. Alternatively, the Src-induced morphological changes may be mediated by activation of Ras (which is constitutively activated in Src- transformed cells due to tyrosine phosphorylation of She (Section 1.17.1.1), PLCy or PI 3-K (161).
Neurite outgrowth by PC 12 cells is also induced by over-expression of Src, suggesting that the kinase is involved in the development of neurones. However, mice which lack Src, Yes and Abl (due to homologous recombination in embryonic stem cells), have no detectable abnormalities in jhippocampal development or impairment of spacial learning and long term potentiation. However, mutations of the related tyrosine kinase, Fyn, affect these processes (162). Mutation of Fyn is accompanied by hypophosphorylation of ppl25™ , which is distributed in the axons and dendrites of neurons, suggesting roles for the tyrosine kinases Fyn and ppl25^^ in brain development and learning. Mutation of other members of the Src family of kinases induces significant increases in the activity of other Src-related kinases. The authors suggest that Src, Yes and Abl are also involved with brain development but that they may be functionally redundant (163). Other studies, using tyrosine kinase inhibitors, have also suggested a role for tyrosine kinases in neurite outgrowth. The length of neurites produced by cerebellar neurons increases when cultured with the tyrosine kinase inhibitor herbimycin A. The effect is inhibited by other tyrosine kinase inhibitors, suggesting that multiple tyrosine kinases regulate neurite outgro^vth (164).
Src appears to be important for signal transduction pathways downstream of receptor tyrosine kinases as dominant inhibitory, kinase-deficient mutants of Src inhibit EGF- and
PDGF-induced mitogenesis. Blockage of PDGF-induced DNA synthesis is rescued by the transcription factor Myc (but not Fos), suggesting that Src may mediate PDGF-induced increased Myc expression, an essential step for entry into the cell cycle (165). Activation of Src may occur by several mechanisms. Phosphorylation of c-Src at residue 527 by the tyrosine kinase Csk inhibits Src kinase activity, possibly due to interactions between the phosphotyrosine and the SH2 domain of Src. Dephosphorylation ofTyrg2 7 or competition with
other phosphotyrosine residues for the Src SH2 domain binding are two alternative mechanisms for Src activation. An autophosphorylation site on the P-PDGF receptor forms a high affinity binding site for the SFI2 domains of c-Src and the related kinases Fyn and Yes, stimulating kinase activity. Alternatively, autophosphorylation of ppl25™ and phosphorylation of paxillin in response to ligand binding to integrin receptors may recruit and activate Src, Fyn and Csk to focal contacts via their SH2 domains. These interactions may be required for mediating signals (from adherence structures) regulating entry into the cell cycle.
Cells derived from mice that lack Csk have changes to the cytoskeleton organization and hyperphosphorylation of several cytoskeletal proteins, including tensin, ppl25^^, paxillin and cortactin, an actin binding protein that is enriched in cortical structures such as membrane ruffles and lamellipodia. Hyperphosphorylation of cortactin and tensin appears to be dependent on Src, since they are not hyperphosphorylated in Src-deficient cells. In contrast, ppl25^^ and paxillin hyperphosphorylation are only partly dependent on Src and Fyn (166).
Abl, a non-receptor tyrosine kinase with a large carboxyl-terminal domain, may regulate actin organization more directly. c-Abl binds F- and G-actin via the carboxyl-terminal domain and is capable of bundling microfilaments in vitro. However, Abl is enriched within the nucleus, with only a proportion associated with the plasma membrane, focal contacts and filamentous structures resembling stress fibres. Fusions between Abl and Bcr (the product of the break point cluster region), resulting from reciprocal translocations between chromosomes 9 and 22, are associated with chronic myelogenous and acute lymphocytic leukaemias, although over-expression of c-Abl results in cell-cycle blockage during the G^ phase, . The function of the actin binding sequences are unclear since they are not required for Abl/Bcr- induced transformations (167).