Abstract
The Src substrate Tks5 helps scaffold matrix-remodeling invadopodia in invasive cancer cells. Focus was directed here on how the five SH3 domains of Tks5 impact that activity. Mutations designed to inhibit protein-protein interactions were created in the individual SH3 domains of Tks5, and the constructs were introduced into the LNCaP prostate carcinoma cell line, a model system with intrinsically low Tks5 expression and which our lab had previously showed the dependence of Src-dependent Tks5 phosphorylation on invadopodia development. In LNCaP cells, acute increases in wild-type Tks5 led to increased gelatin matrix degradation. A similar result was observed when Tks5 was mutated in its 4th or 5th SH3 domains. This was in contrast to the 1st, 2nd, and 3rd SH3 domain mutations of Tks5 where each had a remarkable accentuating effect on gelatin degradation. Conversely, in the invadopodia-competent Src-3T3 model system, mutations in any one of the first three SH3 domains had a dominant negative effect that largely eliminated the presence of invadopodia, inhibited gelatin degradation activity, and redistributed both Src, cortactin, and Tks5 to what are likely endosomal compartments. A hypothesis involving Tks5 conformational states and the regulation of endosomal trafficking is presented as an explanation for these seemingly disparate results.
Highlights
The acquisition of an invasive phenotype among tumor cells can be a turning point in disease trajectory resulting in poorer cancer patient prognosis and increased mortality
Given the success of that assay we considered the utility of this model system in determining the relative contribution of the Src homology 3 (SH3) domains of Tks5 to those processes
Responsible for Tks5 recruitment to invadopodia are the adaptor proteins Nck and Grb2, the former of which utilizes its Src homology 2 (SH2) domain to bind to a phosphotyrosine in Tks5 following Src-dependent Tks5 phosphorylation [11], the latter of which utilizes its SH3 domain to bind to one of Tks5’s proline-rich motifs [13]
Summary
The acquisition of an invasive phenotype among tumor cells can be a turning point in disease trajectory resulting in poorer cancer patient prognosis and increased mortality. Invasive cells exhibit the concerted ability to move through a tissue environment dense in extracellular matrix proteins, including the basement membrane that defines tissue boundaries. In such cases, invasion may require proteolysis of the matrix to open channels for continued motility. There are cytoskeletal structures that aid in the proteolytic invasion of cancer cells called invadopodia [1, 2].
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