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.

It has been shown previously in T cells that stimulation of protein kinase C or the T cell antigen receptor leads to a rapid and persistent activation of p21ras as measured by a dramatic increase in the amount of bound GTP. These stimuli are also known to induce the expression of the T lymphocyte growth factor, interleukin‐2 (IL‐2), an essential growth factor for the immune system. Receptor induced activation of p21ras has been demonstrated in several cell types but involvement of protein kinase C as an upstream activator of p21ras appears to be unique to T cells. In this study we show that p21ras acts as a component of the protein kinase C and T cell antigen receptor downstream signalling pathway controlling IL‐2 gene expression. In the murine T cell line EL4, constitutively active p21ras greatly potentiates the phorbol ester and T cell receptor agonist induced production of IL‐2 as measured both by biological assay for the cytokine and by the use of a reporter construct. Active p21ras also partially replaces the requirement for protein kinase C activation in synergizing with a calcium ionophore to induce production of IL‐2. Furthermore, using a dominant negative mutant of ras, Ha‐rasN17, we show that endogenous ras function is essential for induction of IL‐2 expression in response to protein kinase C or T cell receptor stimulation. Activation of ras proteins is thus a necessary but not sufficient event in the induction of IL‐2 synthesis. Ras proteins are therefore pivotal signalling molecules in T cell activation.

Madin-Darby canine kidney (MDCK) epithelial cells transformed by oncogenic Ras and

SunlmaryIn T lymphocytes, triggering of the T cell receptor (TCR) induces several signaling cascades which ultimately synergize to induce the activity of the nuclear factor of activated T cells (NFAT), a DNA binding complex critical to the inducibility and T cell specificity of the T cell growth factor interleukin 2. One immediate consequence of T cell activation via the TCR is an increase in cytosolic calcium. Calcium signals are important for NFAT induction, and recent studies have identified calcineurin, a calcium-calmodulin dependent serine-threonine phosphatase, as a prominent component of the calcium signaling pathway in T ceils. A second important molecule in TCR signal transduction is the guanine nucleotide binding protein, p21 r~, which is coupled to the TCR by a protein tyrosine kinase dependent mechanism. The experiments presented here show that expression by transfection of mutationally activated calcineurin or activated p21 r~ alone is insuffident for NFAT transactivation. However, coexpression of the activated calcineurin with activated p21 r~ could mimic TCR signals in NFAT induction. These data identify calcineurin and p21 ~as as cooperative partners in T cell activation.

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The aim of this present study was to investigate the role of protein kinase C (PKC), downstream of p21ras, in activating interleukin-2 (IL-2) gene expression. It has been reported that PKC is an effector of p21ras in T cells. Data is presented, using the potent and selective PKC inhibitor Ro 31-8425 and transient expression of a constitutively active ras mutant, which clearly shows that PKC is not downstream of p21ras in the induction of NF-AT and AP-1 transcriptional activity and in the expression of IL-2 in human Jurkat T cells. Reporter gene experiments demonstrated that NF-kappa B transcriptional activity is not affected by expression of activated p21ras. The signaling pathways involving PKC activation, calcium mobilization and ras activation combine to provide the necessary components for production of IL-2 during T cell activation.

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