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The serine/threonine kinase Prk1p is known to be involved in the regulation of the actin cytoskeleton organization in budding yeast. One possible function of Prk1p is the negative regulation of Pan1p, an actin patch regulatory protein that forms a complex in vivo with at least two other proteins, Sla1p and End3p. In this report, we identified Sla1p as another substrate for Prk1p. The phosphorylation of Sla1p by Prk1p was established in vitro with the use of immunoprecipitated Prk1p and in vivo with the use of PRK1 overexpression, and was further supported by the finding that immunoprecipitated Sla1p contained PRK1- and ARK1-dependent kinase activities. Stable complex formation between Prk1p and Sla1p/Pan1p in vivo could be observed once the phosphorylation reaction was blocked by mutation in the catalytic site of Prk1p. Elevation of Prk1p activities in wild-type cells resulted in a number of deficiencies, including those in colocalization of Pan1p and Sla1p, endocytosis, and cell wall morphogenesis, likely attributable to a disintegration of the Pan1p/Sla1p/End3p complex. These results lend a strong support to the model that the phosphorylation of the Pan1p/Sla1p/End3p complex by Prk1p is one of the important mechanisms by which the organization and functions of the actin cytoskeleton are regulated.

Several inducible gene expression systems have been developed in vitro in recent years to overcome limitations with traditional transgenic mice. One of these, the tetracycline-regulated system, has been used successfully in vivo. Nevertheless, concerns remain about the ability of this system to direct high levels of transgene expression in vivo and to enable such expression to be turned on and off effectively. We report here the generation, using a modified tetracycline-regulated system under the control of the neuron-specific enolase promoter, of several lines of mice that direct transgene expression to specific brain regions, including the striatum, cerebellum, CA1 region of the hippocampus, or deep layers of cerebral neocortex. Transgene expression in these mice can be turned off completely with low doses of doxycycline (a tetracycline derivative) and driven to very high levels in the absence of doxycycline. We demonstrate this tissue-specific, inducible expression for three transgenes: those that encode luciferase (a reporter protein) or DeltaFosB or the cAMP-response element binding protein (CREB) (two transcription factors). The various lines of transgenic mice demonstrate an inducible system that generates high levels of transgene expression in specific brain regions and represent novel and powerful tools with which to study the functioning of these (or potentially any other) genes in the brain.