SRChing for the substrates of Src.

By the mid 1980's, it was clear that the transforming activity of oncogenic Src was linked to the activity of its tyrosine kinase domain and attention turned to identifying substrates, the putative next level of control in the pathway to transformation. Among the first to recognize the potential of phosphotyrosine-specific antibodies, Parsons and colleagues launched a risky shotgun-based approach that led ultimately to the cDNA cloning and functional characterization of many of today's best-known Src substrates (for example, p85-Cortactin, p110-AFAP1, p130Cas, p125FAK and p120-catenin). Two decades and over 6000 citations later, the original goals of the project may be seen as secondary to the enormous impact of these protein substrates in many areas of biology. At the request of the editors, this review is not restricted to the current status of the substrates, but reflects also on the anatomy of the project itself and some of the challenges and decisions encountered along the way.

Ableson kinases negatively regulate invadopodia function and invasion in head and neck squamous cell carcinoma by inhibiting an HB-EGF autocrine loop.

Head and neck squamous cell carcinoma (HNSCC) has a proclivity for locoregional invasion. HNSCC mediates invasion in part through invadopodia-based proteolysis of the extracellular matrix (ECM). Activation of Src, Erk1/2, Abl and Arg downstream of epidermal growth factor receptor (EGFR) modulates invadopodia activity through phosphorylation of the actin regulatory protein cortactin. In MDA-MB-231 breast cancer cells, Abl and Arg function downstream of Src to phosphorylate cortactin, promoting invadopodia ECM degradation activity and thus assigning a pro-invasive role for Ableson kinases. We report that Abl kinases have an opposite, negative regulatory role in HNSCC where they suppress invadopodia and tumor invasion. Impairment of Abl expression or Abl kinase activity with imatinib mesylate enhanced HNSCC matrix degradation and 3D collagen invasion, functions that were impaired in MDA-MB-231. HNSCC lines with elevated EGFR and Src activation did not contain increased Abl or Arg kinase activity, suggesting that Src could bypass Abl/Arg to phosphorylate cortactin and promote invadopodia ECM degradation. Src-transformed Abl(-/-)/Arg(-/-) fibroblasts produced ECM degrading invadopodia containing pY421 cortactin, indicating that Abl/Arg are dispensable for invadopodia function in this system. Imatinib-treated HNSCC cells had increased EGFR, Erk1/2 and Src activation, enhancing cortactin pY421 and pS405/418 required for invadopodia function. Imatinib stimulated shedding of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) from HNSCC cells, where soluble HB-EGF enhanced invadopodia ECM degradation in HNSCC but not in MDA-MB-231. HNSCC cells treated with inhibitors of the EGFR-invadopodia pathway indicated that EGFR and Src are required for invadopodia function. Collectively, our results indicate that Abl kinases negatively regulate HNSCC invasive processes through suppression of an HB-EGF autocrine loop responsible for activating a EGFR-Src-cortactin cascade, in contrast to the invasion promoting functions of Abl kinases in breast and other cancer types. Our results provide mechanistic support for recent failed HNSCC clinical trials utilizing imatinib.

Cortactin: coupling membrane dynamics to cortical actin assembly.

Exposure of cells to a variety of external signals causes rapid changes in plasma membrane morphology. Plasma membrane dynamics, including membrane ruffle and microspike formation, fusion or fission of intracellular vesicles, and the spatial organization of transmembrane proteins, is directly controlled by the dynamic reorganization of the underlying actin cytoskeleton. Two members of the Rho family of small GTPases, Cdc42 and Rac, have been well established as mediators of extracellular signaling events that impact cortical actin organization. Actin-based signaling through Cdc42 and Rac ultimately results in activation of the actin-related protein (Arp) 2/3 complex, which promotes the formation of branched actin networks. In addition, the activity of both receptor and non-receptor protein tyrosine kinases along with numerous actin binding proteins works in concert with Arp2/3-mediated actin polymerization in regulating the formation of dynamic cortical actin-associated structures. In this review we discuss the structure and role of the cortical actin binding protein cortactin in Rho GTPase and tyrosine kinase signaling events, with the emphasis on the roles cortactin plays in tyrosine phosphorylation-based signal transduction, regulating cortical actin assembly, transmembrane receptor organization and membrane dynamics. We also consider how aberrant regulation of cortactin levels contributes to tumor cell invasion and metastasis.

Focal adhesion kinase: a regulator of focal adhesion dynamics and cell movement.

Engagement of integrin receptors with extracellular ligands gives rise to the formation of complex multiprotein structures that link the ECM to the cytoplasmic actin cytoskeleton. These adhesive complexes are dynamic, often heterogeneous structures, varying in size and organization. In motile cells, sites of adhesion within filopodia and lamellipodia are relatively small and transient and are referred to as 'focal complexes,' whereas adhesions underlying the body of the cell and localized to the ends of actin stress fibers are referred to as 'focal adhesions'. Signal transduction through focal complexes and focal adhesions has been implicated in the regulation of a number of key cellular processes, including growth factor induced mitogenic signals, cell survival and cell locomotion. The formation and remodeling of focal contacts is a dynamic process under the regulation of protein tyrosine kinases and small GTPases of the Rho family. In this review, we consider the role of the focal complex associated protein tyrosine kinase, Focal Adhesion Kinase (FAK), in the regulation of cell movement with the emphasis on how FAK regulates the flow of signals from the ECM to the actin cytoskeleton.

Regulation of c-SRC activity and function by the adapter protein CAS.

SRC family kinases play essential roles in a variety of cellular functions, including proliferation, survival, differentiation, and apoptosis. The activities of these kinases are regulated by intramolecular interactions and by heterologous binding partners that modulate the transition between active and inactive structural conformations. p130(CAS) (CAS) binds directly to both the SH2 and SH3 domains of c-SRC and therefore has the potential to structurally alter and activate this kinase. In this report, we demonstrate that overexpression of full-length CAS in COS-1 cells induces c-SRC-dependent tyrosine phosphorylation of multiple endogenous cellular proteins. A carboxy-terminal fragment of CAS (CAS-CT), which contains the c-SRC binding site, was sufficient to induce c-SRC-dependent protein tyrosine kinase activity, as measured by tyrosine phosphorylation of cortactin, paxillin, and, to a lesser extent, focal adhesion kinase. A single amino acid substitution located in the binding site for the SRC SH3 domain of CAS-CT disrupted CAS-CT's interaction with c-SRC and inhibited its ability to induce tyrosine phosphorylation of cortactin and paxillin. Murine C3H10T1/2 fibroblasts that expressed elevated levels of tyrosine phosphorylated CAS and c-SRC-CAS complexes exhibited an enhanced ability to form colonies in soft agar and to proliferate in the absence of serum or growth factors. CAS-CT fully substituted for CAS in mediating growth in soft agar but was less effective in promoting serum-independent growth. These data suggest that CAS plays an important role in regulating specific signaling pathways governing cell growth and/or survival, in part through its ability to interact with and modulate the activity of c-SRC.

Beta II-spectrin (fodrin) and beta I epsilon 2-spectrin (muscle) contain NH2- and COOH-terminal membrane association domains (MAD1 and MAD2).

Central to spectrin's function is its association with the plasma membrane. The linking proteins ankyrin and protein 4.1 partly mediate this association, and their interactions with spectrin are well understood. Both beta I (erythrocyte) and beta II (fodrin, beta G) spectrin also associate with unknown protein receptors in crude membrane preparations by ankyrin and protein 4.1 independent mechanisms. As a first step to understanding this interaction, kinetic and equilibrium assays have been used to monitor which regions of beta I and beta II spectrin inhibit the binding of purified 125I-labeled bovine brain spectrin to demyelinated and NaOH-stripped bovine brain membranes. A series of 19 recombinant proteins spanning the entire sequence of beta II spectrin, including an alternatively spliced NH2-terminal isoform (beta II epsilon 2 spectrin), were prepared as glutathione S-transferase fusion proteins. Also prepared were peptides representing the alternatively spliced COOH-terminal domain found in beta I epsilon 2 spectrin ("muscle spectrin"). Two distinct sequence motifs inhibited the binding of native brain spectrin. Membrane association domain 1 (MAD1) was represented in all fusion peptides that included spectrin repeat 1. These peptides slowed the kinetics of brain spectrin binding and inhibited up to 46% of the maximal binding under the conditions of these assays (apparent Ki < or = 0.2 microM). Peptides representative of repeats 2-17 of beta II spectrin were devoid of inhibitory activity. The second membrane association domain (MAD2) was identified in penultimate COOH-terminal sequences (domain III) of both beta II and beta I epsilon 2 spectrin. These sequences were absent in beta I epsilon 1 (erythrocyte) spectrin. MAD2 competitively inhibited over 80% of brain spectrin binding in these assays, with an apparent Ki < or = 0.1 microM. Direct binding studies confirmed that both MAD1 and MAD2 peptides associated with membranes with affinities comparable to their inhibition constants. Sequence comparisons suggest that MAD1 is created by the insertion of two non-homologous sequence motifs into repeat 1, extending it from 106 to 122 amino acids. Similarly, MAD2 encompasses a putative site of beta gamma-heterotrimeric G-protein binding called the pleckstrin homology domain, and MAD2 may in fact be the pleckstrin homology domain although this has not been rigorously proven. Collectively these studies identify two novel functional motifs in spectrin that mediate ankyrin independent association with membranes. We hypothesize that these motifs and their still to be discovered ligands play a primary role in the nascent assembly and stabilization of an ordered and polarized spectrin skeleton.