Abstract
To elucidate the role of focal adhesion kinase (pp125FAK) in transformation, its phosphorylation in transformed fibroblasts was compared with that of detransformed fibroblasts induced by a histone deacetylase inhibitor, trichostatin A (TSA). Inhibition of histone deacetylase activity in two different ras-transformed fibroblast lines by TSA induced a morphological change into a flattened and more spread morphology, implying detransformation. These morphological changes included increased spreading ability of transformed NIH 3T3 cells on fibronectin. Of the six tyrosine phosphorylation sites in pp125FAK, phosphorylation at position 861 (Tyr-861) was clearly decreased during detransformation by TSA. It resulted from decreased activity of Src family tyrosine kinase and/or decreased amount of Src kinase interacting with pp125FAK. Furthermore, phosphorylation of Tyr-861 was reduced substantially by the Src family kinase inhibitor, PP1, while overexpression of Src kinase increased its phosphorylation, implying that Src kinase regulates phosphorylation of pp125FAK at Tyr-861. All of these findings suggest that increased phosphorylation of pp125FAK at Tyr-861 correlates with Ras-induced transformation of fibroblasts, and TSA is able to detransform them through regulation of pp125FAK phosphorylation at Tyr-861 by an Src family kinase.
Highlights
Altered cellular structure, shape, and cytoskeletal architecture are hallmarks of malignant transformation [1]
Since the most clearly changed characteristic is an alteration of cytoskeletal organization, we investigated whether Trichostatin A (TSA) regulates the functions of proteins involved in cytoskeletal organization
The expression of paxillin was slightly increased, whereas other cytoskeletal proteins including vinculin, talin, ␣-actinin, and pp125FAK were unchanged. Since both integrin 1 and RhoA are known as important regulators of ECM-mediated cytoskeletal organization [36], TSA-induced detransformation may be closely related with integrin-mediated adhesion to ECM
Summary
An important mechanism in controlling gene transcription [5, 6]. The acetylation state of histone is regulated by reversible enzymes, histone acetyltransferase and histone deacetylase (HDAC)1 [7,8,9]. Association of c-Src leads to additional pp125FAK phosphorylation at Tyr-925 to create binding sites for SH2-containing proteins such as growth factor receptor-bound protein 2 (Grb2) [26, 31], which in turn activates the ras cascade pathway. Similar to transformed cells, increased metastatic activity of prostate cancer cell lines correlates with increased pp125FAK expression and increased overall tyrosine phosphorylation [33]. Inhibition of HDAC activity down-regulates Src kinase activity, and in turn decreases phosphorylation of pp125FAK at Tyr-861. These changes, together with increased expression of several cytoskeletal proteins, result in spreading of Ha-ras-transformed NIH3T3 cells with a detransformed morphology. TSA-induced detransformation correlates with decreased pp125FAK phosphorylation at tyrosine 861 in ras-transformed fibroblasts
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