Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
1998-03-03
2002-08-20
Crouch, Deborah (Department: 1632)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S004000
Reexamination Certificate
active
06436656
ABSTRACT:
TECHNICAL FIELD
The present invention relates, in general, to immunosuppression and, in particular, to a method of inducing an immunosuppressive effect. The invention further relates to a method of screening compounds for immunosuppressive activity.
BACKGROUND
Ca
2+
/calmodulin-dependent protein kinase IV (CaMKIV) is a monomeric multifunctional enzyme that is expressed only in subanatomical portions of the brain, T lymphocytes and postmeiotic male germ cells. It is present in the nucleus of cells in which it is expressed (Jensen et al, Proc. Natl. Acad. Sci. USA 88:2850 (1991)). CaMKIV phosphorylates and activates the cyclic AMP response element binding proteins CREB and CREM&tgr; in a manner analogous to protein kinase A (Matthews et al, Mol. Cell. Biol. 14:6107 (1994); Sun et al, Genes Dev. 8:2527 (1994); Enslen et al, J. Biol. Chem. 269:15320 (1994)).
In the absence of Ca
2+
/calmodulin, CaMKIV is inactive. Activation requires three events: i) binding of Ca
2+
/calmodulin; ii) phosphorylation of a single threonine residue present in the activation loop by a separate protein kinase that is also Ca
2+
/calmodulin-dependent; and iii) autophosphorylation of serine residues present in the extreme N-terminus that is required to relieve a novel form of autoinhibition.
The gene for rat CaMKIV has been cloned and found to span 42 kb of DNA. The gene encodes
3
proteins namely, the &agr; and &bgr; forms of CaMKIV that differ only in that the &bgr; form contains a 28 amino acid N-terminal extension as well as calspermin (Sun et al, J. Biol. Chem. 270:29507 (1995)). Calspermin is the C-terminal 169 amino acids of CaMKIV that binds Ca
2+
/calmodulin and is expressed only in posmeiotic male germ cells. The promoter for calspermin resides in the penultimate intron of the CaMKIV gene and is regulated by two CREs. Available data suggest that rearrangement of chromatin during meiosis together with the expression of CREM&tgr; at high levels are sufficient to control expression of the calspermin promoter during spermatogenesis. On the other hand, the developmental expression of CaMKIV in cells of the brain and T cells appears to be controlled by thyroid hormone mediated via the thyroid hormone receptor &agr;.
Prior to the present invention, the specific role of CaMKIV in T cell activation had not been defined and the potential importance of CaMKIV as a target for immunosuppressive agents had not been suggested. Indeed, currently available immunosuppressive drugs, as well as new generation drugs presently under development, target calcineurin. Since calcineurin is present in all cells, not just T cells, these drugs are associated with a plethora of side effects. The present invention provides a method of identifying immunosuppressive agents that target CaMKIV and that are T cell specific and thus substantially free of adverse side effects.
OBJECTS AND SUMMARY OF THE INVENTION
It is one object of the invention to provide a method of inducing an immunosuppressive effect, while avoiding undesirable side effects.
It is another object of the invention to provide a method of screening compounds for immunosuppressive activity.
It is a further object of the invention to provide a method of inhibiting CREB phosphorylation specifically in T cells.
The foregoing objects are met by the present invention which results, at least in part, from the recognition that inhibition of CaMKIV prevents phosphorylation of CREB and thereby activation of T cells.
Further objects and advantages of the present invention will be clear from the description that follows.
REFERENCES:
Kitani et al., “Inactivation of Ca2+/Calmodulin-dependent protein kinase IV by Ca2+/Calmodulin and restoration of the activity by Mg2+/EGTA”, J. Biochem., 117(5):1070-1075, May 1995.*
Park et al., “Activation of Ca2+/Calmodulin-dependent protein kinase (CaM-kinase) IV by CaM-kinase kinase in Jurkat T lymphocytes”, J. Biol. Chem., 270(51):30464-30469, Dec. 1995.*
Chatila et al., “A unique phosporylation-dependent mechanism for the activation of Ca2+/Calmodulin-dependent protein type IV/GR”, J. Biol. Chem., 271(35):21542-21548, Aug. 1996.*
Chatila et al, “A Unique Phosphorylation-dependent Mechanism for the Activation of Ca2+/Calmodulin-dependent Protein Kinase Type IV/GR”, The Journal of Biological Chemistry 271(35):21542-21548 (1996).
Tokumitsu et al, “Activation Mechanisms for Ca2+/Calmodulin-dependent Protein Kinase IV”, The Journal of Biological Chemistry 269(46):28640-28647 (1994).
Sikela et al, “Chromosal Localization of the Human Gene for Brain Ca2+Calmodulin-Dependent Proten Kinase Type IV”, Genomics 4:21-27 (1989).
Sun et al, “Organzation and Analysis of the Complete Rat Calmodulin-dependent Protein Kinase IV Gene”, The Journal of Biological Chemistry 270(49):29507-29514 (1995).
Sikela et al, “Genetic Mappying of the Gene for Ca2+/Calmodulin-Dependent Protein Kinase IV (Camk-4) to Mouse Chromosome 18”, Genomics 8-579-582 (1990).
Jones et al, “cDNA sequence and differential expression of the mouse Ca2+/calmodulin-dependent protein kinase IV gene”, FEBS Letters 289(1):105-109 (1991).
Okuno et al, “Evidence for the Existence of Ca2+/Calmodulin-Dependent Protein Kinase IV Kinase Isoforms in Rat Brain”, J. Biochem. 119-1176-1181 (1996).
Tokumitsu et al, “Characterization of a Ca2+/Calmodulin-dependent Protein Kinase Cascade”, The Journal of Biological Chemistry 270(33):19320-19324 (1995).
Enslen et al, “Characterization of Ca2+/Calmodulin-dependent Protein Kinase IV”, The Journal of Biological Chemistry 269(22):15520-15527 (1994).
Sakagami and Kondo, “Cloning and sequencing of a gene encoding the &bgr; polypeptide of Ca2+/calmodulin-dependent protein kinase IV and its expression confined to the mature cerebellar granule cells”, Molecular Brain Research 19:215-218 (1993).
Kitani et al, “Inactivation of Ca2+/Calmodulin-Dependent Protein Kinase IV by Ca2+/Calmodulin and Restoration of the Activity by Mg2+/EGTA”, J. Biochem. 117:1070-1075 (1995).
Butler et al, “Limbic epilepsy in transgenic mice carrying a Ca2+/calmodulin-dependent kinase II &agr;-subunit mutation”, Proc. Natl. Acad. Sci. USA 92:6852-6855 (1995).
Crouch Deborah
Duke University
Nixon & Vanderhye P.C.
Woitach Joseph T.
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