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Tks5/Fish is a scaffolding protein with five SH3 domains and one PX domain. In Src-transformed cells, Tks5/Fish localizes to podosomes, discrete protrusions of the ventral membrane. We generated Src-transformed cells with reduced Tks5/Fish levels. They no longer formed podosomes, did not degrade gelatin, and were poorly invasive. We detected Tks5/Fish expression in podosomes in invasive cancer cells, as well as in human breast cancer and melanoma samples. Tks5/Fish expression was also required for protease-driven matrigel invasion in human cancer cells. Finally, coexpression of Tks5/Fish and Src in epithelial cells resulted in the appearance of podosomes. Thus, Tks5/Fish appears to be required for podosome formation, for degradation of the extracellular matrix, and for invasion of some cancer cells.

Fish is a scaffolding protein and Src substrate. It contains an amino-terminal Phox homology (PX) domain and five Src homology 3 (SH3) domains, as well as multiple motifs for binding both SH2 and SH3 domaincontaining proteins. We have determined that the PX domain of Fish binds 3-phosphorylated phosphatidylinositols (including phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate). Consistent with this, a fusion protein of green fluorescent protein and the Fish PX domain localized to punctate structures similar to endosomes in normal fibroblasts. However, the full-length Fish protein was largely cytoplasmic, suggesting that its PX domain may not be able to make intermolecular interactions in unstimulated cells. In Src-transformed cells, we observed a dramatic re-localization of some Fish molecules to actin-rich structures called podosomes; the PX domain was both necessary and sufficient to effect this translocation. We used a phage display screen with the fifth SH3 domain of Fish and isolated ADAM19 as a binding partner. Subsequent analyses in mammalian cells demonstrated that Fish interacts with several members of the ADAMs family, including ADAMs 12, 15, and 19. In Src-transformed cells, ADAM12 co-localized with Fish in podosomes. Because members of the ADAMs family have been implicated in growth factor processing, as well as cell adhesion and motility, Fish could be acting as an adaptor molecule that allows Src to impinge on these processes.

The homotypic fusion of yeast vacuoles requires Sec18p (NSF)-driven priming to allow vacuole docking, but the mechanism that links priming and docking is unknown. We find that a large multisubunit protein called the Vam2/6p complex is bound to cis-paired SNAP receptors (SNAREs) on isolated vacuoles. This association of the Vam2/6p complex with the cis-SNARE complex is disrupted during priming. The Vam2/6p complex then binds to Ypt7p, a guanosine triphosphate binding protein of the Rab family, to initiate productive contact between vacuoles. Thus, cis-SNARE complexes can contain Rab/Ypt effectors, and these effectors can be mobilized by NSF/Sec18p-driven priming, allowing their direct association with a Rab/Ypt protein to activate docking.

A vacuole membrane-associated calcium-binding protein with an apparent mass of 45 kD was purified from celery (Apium graveolens). This protein, VCaB45, is enriched in highly vacuolate tissues and is located within the lumen of vacuoles. Antigenically related proteins are present in many dicotyledonous plants. VCaB45 contains significant amino acid identity with the dehydrin family signature motif, is antigenically related to dehydrins, and has a variety of biochemical properties similar to dehydrins. VCaB45 migrates anomalously in sodium dodecyl sulfate-polyacrylamide gel electrophoresis having an apparent molecular mass of 45 kD. The true mass as determined by matrix-assisted laser-desorption ionization time of flight was 16.45 kD. VCaB45 has two characteristic dissociation constants for calcium of 0.22 Ϯ 0.142 mm and 0.64 Ϯ 0.08 mm, and has an estimated 24.7 Ϯ 11.7 calcium-binding sites per protein. The calcium-binding properties of VCaB45 are modulated by phosphorylation; the phosphorylated protein binds up to 100-fold more calcium than the dephosphorylated protein. VCaB45 is an "in vitro" substrate of casein kinase II (a ubiquitous eukaryotic kinase), the phosphorylation resulting in a partial activation of calcium-binding activity. The vacuole localization, calcium binding, and phosphorylation of VCaB45 suggest potential functions.The vacuole is a reservoir for calcium (Machlon, 1984) and consequently plays an important role in calcium homeostasis (Miller et al., 1990;Allen and Sanders, 1995;Sanders et al., 1999). Regulation of vacuole calcium levels is complex involving a variety of calcium channels and pumps (Sanders et al., 1999;Sze et al., 2000). Sustained elevated levels of cytosolic calcium can be toxic (Hepler and Wayne, 1985), so under normal conditions, cytosolic calcium levels increase only transiently. Proteinaceous calcium buffers may serve as homeostats to attenuate the signal transduction system. Well-characterized protein calcium buffers include calreticulin and calsequestrin (Ostwald and MacLennon, 1974;Campbell et al., 1983b). Homologs of calsequestrin (Krause et al., 1989;Xing et al., 1994), calreticulin (Chen et al., 1994;Napier et al., 1995;Nelson et al., 1997), and calnexin (Li et al., 1998) have been identified in plants. These calcium-binding proteins can bind on the order of 20 to 50 calcium ions with both high-(1-3 sites per protein) and low-(20-50 sites per protein) affinity sites. The levels of calcium binding proteins may have a significant impact on signaling processes and may regulate second messenger transmission (Camacho and Lechleiter, 1995;Mery et al., 1996;Coppolino et al., 1997). In an alternative role, calcium-dependent interactions of calnexin and calreticulin have been characterized with a variety of proteins (Nigam et al., 1994;Peterson et al., 1995) and both are implicated in the promotion of correct protein folding (Hebert et al., 1996). These latter activities clearly suggest a molecular chaperone role. Recently, a high-capacity, low-affinity calcium-binding protei...

Podosomes and invadopodia are electron-dense, actin-rich protrusions located on the ventral side of the cellular membrane. They are detected in various types of normal cells, but also in human cancer cells and in Src-transformed fibroblasts. Previously we have shown that the scaffold protein Tks5 (tyrosine kinase substrate 5) co-localizes to podosomes/invadopodia in different human cancer cells and in Src-transformed NIH-3T3 cells. Upon reduced expression of Tks5 podosome formation is decreased, which leads to diminished gelatin degradation in vitro in various human cancer cell lines. It is unclear, however, whether cancer cells need podosomes for tumor growth and metastasis in vivo. To test this idea, we evaluated the ability of Srctransformed NIH-3T3 cells, showing stable reduced expression of Tks5 and podosome formation (Tks5 KD), to form subcutaneous tumors in mice. We demonstrate that decreased expression of Tks5 correlated with reduced tumor growth at this site. In addition, we generated lung metastases from these cells following tail vein injection. The lungs of mice injected i.v. with the Tks5 KD showed smaller-sized metastases, but there was no difference in the number of lesions compared to the controls, indicating that podosomes may not be required for extravasation from the blood stream into the lung parenchyma. Independent of the microenvironment however, the reduced tumor growth correlated with decreased tumor vascularization. Our data potentially implicate a novel role of podosomes as mediators of tumor angiogenesis and support further exploration of how podosome formation and Tks5 expression contribute to tumor progression.

The study of tumor viruses has contributed much to our understanding of the mechanisms by which cancer cells arise, resist signals that restrain the growth of normal cells, and metastasize. The study of the oncogene src has been particularly informative. Src-transformed cells are morphologically transformed and highly invasive both in vitro and in vivo. The src gene product is a membrane-associated protein tyrosine kinase. Since the intrinsic catalytic activity of Src is absolutely required for transformation, identifying and studying its substrates has proved invaluable in understanding many aspects of the cancer phenotype, including factor-independent growth, motility, and escape from apoptosis (Martin 2001;Frame 2002).Several years ago, we developed a screen to rapidly isolate Src substrates, regardless of their abundance or ability to associate with Src (Lock et al. 1998). This involved using an enriched preparation of Src to phosphorylate proteins produced from phage expressing mammalian cDNA libraries. Positive clones were identified by immunoblotting with anti-phosphotyrosine antibodies. Our screen was validated by the cloning of many established Src substrates. We also implicated several known proteins as Src substrates, and we cloned several novel cDNAs (Courtneidge 2003). Further analysis of each of these novel clones in mammalian cells confirmed that they were indeed Src substrates. One of these substrates, Tks5, is the subject of our current research and is described here. Tks5: A Src SUBSTRATE AND SCAFFOLD PROTEINThe partial cDNAs we isolated from the Src substrate screen were designated Tks, for tyrosine kinase substrate. Conceptual translation of one such clone, Tks5, showed that it contained several possible Src phosphorylation sites and part of an SH3 domain (Lock et al. 1998). A fulllength clone of Tks5 was then isolated and sequenced.Two distinct Tks5 transcripts were identified in a mouse embryonic cDNA library. Each transcript encodes a protein with an amino-terminal phox homology (PX) domain, five SH3 domains, and multiple proline-rich motifs. The larger transcript differs from the smaller by the presence of two alternative splices, positioned each side of SH3#1 (Fig. 1). We have yet to explore fully the utilization of these transcripts, although our preliminary data suggest that fibroblasts in culture predominantly express a form of Tks5 containing only the second alternatively spliced exon. No catalytic domain was found in the protein, and it is therefore designated as a scaffolding or adapter protein, whose function is to interact with other proteins and lipids (see below).We initially named the protein encoded by this locus Fish (for five SH3 domains). However, because of the potential for confusion, and the difficulty this name creates when searching databases, we have now reverted to the designation Tks5. Note also that in many databases, the gene encoding Tks5 has been given the name SH3MD1.At the time that we originally cloned Tks5, which is located on human chromosome 10, we found ...