GENETIC AND BIOCHEMICAL ANALYSIS OF CELL BEHAVIOR

点击翻译按钮获取中文摘要

Cyclic adenosine-5'-monophosphate (cAMP) plays an important role in cell differentiation and malignant transformation. We have previously shown by generating a series of mutants of Chinese hamster ovary (CHO) cells that virtually all of the effects of cAMP on CHO cells are mediated through cAMP-dependent protein kinase. Dominant negative mutants affecting the regulatory (RI) subunits of this kinase are easily obtained in this system, since holoenzyme cAMP-dependent protein kinase normally consists of two RI and two catalytic (C) subunits in which the activity of the C subunits is inhibited by RI. Any mutation which blocks the ability of RI to interact with cAMP, thereby inhibiting the ability of cAMP to dissociate the complex, which releases active C, or which increases the association of RI and C will act as a dominant negative mutant and block cAMP effects. A similar ablation of the effect of cAMP can be achieved with vectors which allow overexpression of yeast phosphodiesterase, which degrades cAMP as it is formed. To study the interaction of RI with C we have expressed the Chinese hamster recombinant RI and C proteins in E. coli and demonstrated that active, interactive subunits can be synthesized in this system. A variety of mutations which affect cAMP binding to RI and interaction of RI with C have been characterized in this system. In addition, we have discovered that co-expression of RI and C in E. coli results in growth inhibition of the bacteria. This growth inhibition requires functional RI and active C as well as cAMP, and hence mimics cAMP effects in mammalian cells. We have used this system to begin to isolate mutants of RI with the goal of generating a detailed map of functional and non-functional regions of this important regulatory protein. Furthermore, we have developed an expression system for mutant RIs which allows direct selection of dominant negative RI mutants in mammalian cells. Finally, efforts to overexpress yeast phosphodiesterase in transgenic mice have led to an unexpected finding: one of our transgenic mice is severely immunodeficient when bred as a homozygote, suggesting insertional inactivation of a gene essential for normal development of the immune system.