Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving viable micro-organism
Patent
1995-12-14
1999-09-28
Elliott, George C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving viable micro-organism
4352542, 43525421, C12N 119, C12Q 102
Patent
active
059587210
DESCRIPTION:
BRIEF SUMMARY
This invention relates to the screening of candidate substances for potential as pharmaceutical agents. More particularly, it provides a method by which test substances can be screened for their ability to affect a MAP kinase pathway in mammals. Methods are provided for screening test substances for inhibition or activation of the pathway. The invention also provides yeast which are of use in the methods.
It is well known that pharmaceutical research leading to the identification of a new drug generally involves the screening of very large numbers of candidate substances, both before, and even after, a lead compound has been found. This is one factor which makes pharmaceutical research very expensive and time-consuming, so that a method for assisting in the screening process can have considerable commercial importance and utility.
In mammalian cells the activation of the enzyme MAP kinase (MAPK) is a consequence of growth factor stimulation, and is a requirement for cell proliferation (61). Since oncogenic p21 ras proteins transform cells, and inhibition of the normal p21 ras proteins in cells interferes with growth factor signalling, it has been generally assumed that these proteins are involved in the control of cell proliferation. In particular it appears that they are involved in transmitting signals from growth factor receptors to cytoplasmic signal transduction pathways, since both tyrosine kinase-type growth factor receptors and non-tyrosine kinase growth factor receptors require normal p21 ras functions to stimulate MAPK activity and cell proliferation. It seems, therefore, that oncogenic forms of p21 ras uncouple the activation of MAPK from the requirement for external growth factor signals.
It has also been found that the activation of intracellular protein kinase C (PKC) by phorbol esters stimulates MAPK activity without normal ras function in some cell types. It has further been shown that oncogenic p21 Ras introduced into quiescent 3T3 cells rapidly activates PKC and leads to the activation of MAPK in the absence of any external stimuli.
It seemed to us that the activation of MAPK from ras or PKC proceeds successively via the Raf protein kinase and MAPK kinase (MAPKK), essentially along the lines: ##STR1##
It should be noted that there is a family of MAP-kinases and that the kinase have been purified from fibroblasts with molecular weights Activation requires an ordered phosphorylation of a threonine and tyrosine
Yeast MAPK-pathway homologue proteins are involved in yeast signal transduction, including in response to mating pheromones. In the case of the yeast Schizosaccharomyces pombe one MAPK protein is Spk1. Two additional kinases, Byr1 and Byr2, lie in the same pathway as Spk1, of which Byr1 has been shown to have some sequence homology to MAPKK. In addition, the mating pheromone pathway in Spk1 requires Ras protein function, and Byr1 and Byr2 are thought to act downstream of Ras in this pathway. It is possible, therefore that the way in which ras is coupled to these kinase cascades is similar in fission yeast and higher eukaryotes. More particularly, we believe a pathway in S. pombe to be essentially of the form:
There are equivalent proteins in as follows: kinase homologues and components with equivalent function to those in the mammlian MAPK pathway: the HOG1 and MPK1 pathways. MPK1 is a yeast MAPK and has the following components in its pathway:
In all cases, various additional components may act upstream in response to a stimulus, which may come from outside the organism.
Surprisingly, when we placed a mammalian (human) Raf, or a deletional derivative thereof, together with MAPKK, in a yeast strain deficient in either byr1 or byr2 , the engineered strain would mate, indicating that the pathway was functioning, while expression of raf or the raf derivative alone or MAPKK alone did not allow byr1 or byr2 mutant cells to mate. This strongly suggests that Raf can directly phosphorylate and activate MAPKK. It also suggested to us the replacement of spk1 with MAPK and/or yeast ras with mamm
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Ashworth Alan
Hughes David Anthony
Marshall Christopher John
Cancer Research Campaign Technology Ltd.
Elliott George C.
Larson Thomas G.
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