Assays for protein kinases using fluorescent protein substrates

Details Assays for protein kinases using fluorescent protein substrates Assays for protein kinases using fluorescent protein substrates

1999-03-05

2001-06-19

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving transferase

C435S194000

Reexamination Certificate

active

06248550

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to the field of enzymatic assays and, in particular, assays for protein kinase activity involving modified fluorescent proteins.
Protein phosphorylation is one of the most important general mechanisms of cellular regulation. Protein phosphorylation commonly occurs on three major amino acids, tyrosine, serine or threonine, and changes in the phosphorylation state of these amino acids within proteins can regulate many aspects of cellular metabolism, regulation, growth and differentiation. Changes in the phosphorylation state of proteins, mediated through phosphorylation by kinases, or dephosphorylation by phosphatases, is a common mechanism through which cell surface signaling pathways transmit and integrate information into the nucleus. Given their key role in cellular regulation, it is not surprising that defects in protein kinases and phosphatases have been implicated in many disease states and conditions. For example, the over-expression of cellular tyrosine kinases such as the EGF or PDGF receptors, or the mutation of tyrosine kinases to produce constitutively active forms (oncogenes) occurs in many cancer cells. Drucker et al. (1996)
Nature Medicine
2: 561-56. Protein tyrosine kinases are also implicated in inflammatory signals. Defective Thr/Ser kinase genes have been demonstrated to be implicated in several diseases such as myotonic dystrophy as well as cancer, and Alzheimer's disease (Sanpei et al. (1995)
Biochem. Biophys. Res. Commun.
212: 341-6; Sperber et al (1995)
Neurosci. Lett.
197: 149-153; Grammas et al (1995)
Neurobiology of Aging
16: 563-569; Govoni et al. (1996)
Ann. N. Y. Acad. Sci.
777: 332-337).
The involvement of protein kinases and phosphatases in disease states makes them attractive targets for the therapeutic intervention of drugs, and in fact many clinically useful drugs act on protein kinases or phosphatases. Examples include cyclosporin A which is a potent immunosuppressant that binds to cyclophilin. This complex binds to the Ca/calmodulin-dependent protein phosphatase type 2B (calcineurin) inhibiting its activity, and hence the activation of T-cells. (Sigal and Dumont (1992), Schreiber and Crabtree (1992)). Inhibitors of protein kinase C are in clinical trails as therapeutic agents for the treatment of cancer. (
Clin. Cancer Res.
(1995) 1:113-122) as are inhibitors of cyclin dependent kinase. (
J. Mol. Med.
(1995) 73:(10):509-14.)
The number of known kinases and phosphatases are growing rapidly as the influence of genomic programs to identify the molecular basis for diseases have increased in size and scope. These studies are likely to implicate many more kinase and phosphatase genes in the development and propagation of diseases in the future, thereby making them attractive targets for drug discovery. However current methods of measuring protein phosphorylation have many disadvantages which prevents or limits the ability to rapidly screen using miniaturized automated formats of many thousands of compounds. This is because current methods rely on the incorporation and measurement of
32
P into the protein substrates of interest. In whole cells this necessitates the use of high levels of radioactivity to efficiently label the cellular ATP pool and to ensure that the target protein is efficiently labeled with radioactivity. After incubation with test drugs, the cells must be lysed and the protein of interest purified to determine its relative degree of phosphorylation. This method requires high numbers of cells, long preincubation times, careful manipulation and washing steps (to avoid artifactual phosphorylation or dephosphorylation), as well as a method of purification of the target protein. Furthermore, final radioactive incorporation into target proteins is usually very low, giving the assay poor sensitivity. Alternative assay methods, for example based on phosphorylation-specific antibodies using ELISA-type approaches, involve the difficulty of producing antibodies that distinguish between phosphorylated and non-phosphorylated proteins, and the requirement for cell lysis, multiple incubation and washing stages which are time consuming, complex to automate and potentially susceptible to artifacts.
Kinase assays based on purified enzymes require large amounts of purified kinases, high levels of radioactivity, and methods of purification of the substrate protein away from incorporated
32
P-labelled ATP. They also suffer from the disadvantage of lacking the physiological context of the cell, preventing a direct assessment of a drugs toxicity and ability to cross the cells plasma membrane.
Fluorescent molecules are attractive as reporter molecules in many assay systems because of their high sensitivity and ease of quantification. Recently, fluorescent proteins have been the focus of much attention because they can be produced in vivo by biological systems, and can be used to trace intracellular events without the need to be introduced into the cell through microinjection or permeabilization. The green fluorescent protein of
Aequorea victoria
is particularly interesting as a fluorescent indicator protein. A cDNA for the protein has been cloned. (D. C. Prasher et al., “Primary structure of the
Aequorea victoria
green-fluorescent protein,”
Gene
(1992) 111:229-33.) Not only can the primary amino acid sequence of the protein be expressed from the cDNA, but the expressed protein can fluoresce. This indicates that the protein can undergo the cyclization and oxidation believed to be necessary for fluorescence. The fluorescence of green fluorescent protein is generated from residues S65-Y66-G67.
Fluorescent proteins have been used as markers of gene expression, tracers of cell lineage and as fusion tags to monitor protein localization within living cells. (M. Chalfie et al., “Green fluorescent protein as a marker for gene expression,”
Science
263:802-805; A. B. Cubitt et al., “Understanding, improving and using green fluorescent proteins,”
TIBS
Nov. 20, 1995, pp. 448-455. U.S. Pat. No. 5,491,084, M. Chalfie and D. Prasher. Furthermore, mutant versions of green fluorescent protein have been identified that exhibit altered fluorescence characteristics, including altered excitation and emission maxima, as well as excitation and emission spectra of different shapes. (R. Heim et al., “Wavelength mutations and posttranslational autoxidation of green fluorescent protein,”
Proc. Natl. Acad. Sci. USA,
(1994) 91:12501-04; R. Heim et al., “Improved green fluorescence,”
Nature
(1995) 373:663-665.) These properties add variety and utility to the arsenal of biologically based fluorescent indicators.
There is a need for assays of protein phosphorylation that are simple, sensitive, non-invasive, applicable to living cells and tissues and that avoid the use of any radioactivity.
SUMMARY OF THE INVENTION
When fluorescent proteins are modified to incorporate a phosphorylation site recognized by a protein kinase, the fluorescent proteins not only can become phosphorylated by the protein kinase, but they also can exhibit different fluorescent characteristics in their un-phosphorylated and phosphorylated forms when irradiated with light having a wavelength within their excitation spectrum. This characteristic makes fluorescent protein substrates particularly useful for assaying protein kinase activity in a sample.
This invention provides methods for determining whether a sample contains protein kinase activity. The methods involve contacting the sample with a phosphate donor, usually ATP, and a fluorescent protein substrate of the invention; exciting the fluorescent protein substrate with light of an appropriate wavelength; and measuring the amount of a fluorescent property that differs in the un-phosphorylated state and phosphorylated state. An amount that is consistent with the presence of the fluorescent protein substrate in its phosphorylated state indicates the presence of protein kinase activity, and an amount that is consistent with the presence of the protein substrate in its un-phosphoryla

Affiliated with

Also associated with

Rating

Assays for protein kinases using fluorescent protein substrates does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Assays for protein kinases using fluorescent protein substrates, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Assays for protein kinases using fluorescent protein substrates will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2471307