Protein/nucleic Acid Interactions In Vertebrate Embryogenesis

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The NC plays a critical role in the developmental of the vertebrate head, face and jaws, providing the bulk of the craniofacial skeleton as well as peripheral nervous system and other cranial tissues. Normal craniofacial development depends on proper induction, migration and differentiation of NC cells and derivatives. Deficiencies at any of these steps, whether due to intrinsic defects in NC itself, or in failure of NC cells to interact properly with adjacent tissues, can lead to birth defects: up to a third of all congenital malformations are craniofacial in nature. In this project we have used a key NC control factor, TFAP2, as a molecular tool to identify target genes encoding proteins that are required for NC migration and differentiation into craniofacial tissue. The three genes that comprise the focus of this research are either themselves novel (PCNS and Inka) or have not previously been shown to function in NC (MyoX), and have an excellent potential to yield new insights into craniofacial development. In particular, our findings with Inka promise to lead to a new link between cytoskeletal regulation through small GTPases and PAK4 and regulation of gene expression through modulation of cell-cell signaling. In the previous two years we completed and published our studies on PCNS and MyoX, and now focus on focus on Inka and its interaction partners in cells and embryos. Using yeast two-hybrid screening we discovered that Inka interacts with p21-activated kinase 4 (PAK4). This kinase is part of a family of proteins that mediate cell-cell signaling through the small GTPase molecules Rac, Rho and Cdc42. We have found that stable expression of mouse Inca in NIH3T3 cells results in inhibition of PAK4 phosphorylation. PAK4 has been reported to bind to actin filaments and regulate their dynamic remodeling through LIM kinase 1 and cofilin. If Inka controls PAK4 activity, this would predict an effect on the microfilament network in cells. We have tested and confirmed this hypothesis by examining phalloidin staining of a stable Inca expressing NIH3T3 line and showing that these cells have enhanced stress fiber and focal adhesion networks, and also migrate more rapidly than controls. Rescue/reversion experiments showed that these functions depend on the kinase activity of PAK4. Several lines of evidence link the regulation of microfilaments and microtubules, suggesting the possibility that Inka might also affect microtubule dynamics, either indirectly by altering microfilaments or by a more direct mechanism. This was also investigated in the Inca expressing cell lines, and also in Xenopus embryos. We discovered that the level of alpha-tubulin acetylation is inhibited by Inka. This also appears to be mediated via PAK4, but via a mechanism that is independent of the kinase activity. The inhibition appears to be exerted at the level of acetylation, rather than the deacetylation step that has been characterized by other laboratories. Interestingly, Inka also inhibits the acetylation of histones, suggesting a possible role in the control of chromatin architecture. Such a molecular link between the regulation of gene expression and cytoskeletal dynamics would be represent a novel mechanism for coordinating cell behavior and differentiation.