SCREENING FOR COMPOUNDS THAT INHIBIT PHOSPHORYLATION OF I kappa B alpha

Abstract: A method to identify a compound that inhibits the phosphorylation of residues serine 32 and serine 36 of I kappa B alpha by CKII is described. A method to identify a compound that selectively inhibits the up-regulation of CKII is also described.

Description: SCREENING FOR COMPOUNDS THAT INHIBIT PHOSPHORYLATION OF IKBC. Statement as to Federally Sponsored Research Funding for the work described herein was provided by the federal government, which may have certain rights in the invention. Background of the Invention Nuclear factor kappa B (NF-κB) is a prototypic transcription factor involved in the regulation of numerous genes. Classic NF-/B heterodimers are composed of p50 (NF-KBI) and p65 (RelA) subunits and make up a significant portion of the inducible pool of NF-κB anchored in the cytosol through the association of p65 with inhibitory molecules known as IKBS. Barnes, P.J., Int. J. Biochem. Cell Biol . , 29 (6) : 867-870 (1997). These include I/cBα, ε, and the precursor proteins of p50 and p52, pl05 and plOO, respectively. I/cBα, the most well studied of these inhibitors, is a 35-37 kD protein that contains six ankyrin repeats flanked by a PEST region in the acidic C-terminus. The N-terminal domain of IκBα contains key amino acids known to participate in stimuli - induced degradation of I/cBc. by tumor necrosis factor (TNF), phorbol 12-myristate 13-acetate (PMA) -ionomycin and lipopolysaccharide (LPS) . The basal half-life of I/cBce is relatively short (30-180 minutes) with cellular activation by a variety of stimuli leading to an even more rapid IKBQ; degradation, ultimately allowing for the release and translocation of large pools of NF-κB into the nucleus. The mechanisms controlling the stimulus- induced degradation and the basal turnover of IκBα are being investigated with a major emphasis on studying the phosphorylation modifications that appear to govern these two processes. Summary of the Invention The present invention relates to the ability of casein-kinase II (CKII) to phosphorylate residues serine 32 and serine 36 of I κBoι , inducing I κ.Ba degradation and consequently, activating NF-«B. This phosphorylation activity of CKII is inducible by HIV and tumor promoters such as PMA. The invention features a method to identify a compound that inhibits the phosphorylation of residues serine 32 and serine 36 of IκBα by CKII. The method includes incubating an IKBC. polypeptide and CKII with a compound and determining if phosphorylation of residues serine 32 and serine 36 is inhibited. An I κB polypeptide that is a C-terminal truncation of full- length I KBOI is particularly useful in the method. CKII is used herein interchangeably with "protein-kinase CKII" (PK-CKII) and refers to the holoenzyme, a catalytic subunit (s), or a biologically active fragment of a catalytic subunit. CKII can consist essentially of the alpha and alpha' catalytic subunits, and in particular, the alpha subunit of the CKII holoenzyme, and can be from HIV infected cells. The invention also relates to a method for identifying candidate compounds selectively inhibiting the up-regulation of CKII. The method includes contacting a cell preparation with a compound and monitoring the phosphorylation of residues serine 32 and serine 36 of I κ.Ba polypeptide. An I κBa polypeptide that is a C-terminal truncation of full-length I κBa is particularly useful in this method. CKII can consist essentially of the catalytic alpha and alpha' subunits, and in particular, the alpha subunit of the CKII holoenzyme. The cell preparation can include viable cells or cellular lysates, and can be from HIV infected cells, malignant cells, cells exposed to a tumor promoter such as PMA. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. Brief Description of the Drawing Figure 1 is an autoradiogram depicting that PK- CKII inhibitors block the slower migrating form of I κBa . Lane 1 is in the presence of [ 35 S] -labelled wild type I KBOI (wt) . Lanes 2-4 are in the presence of wt I κBa and cell extracts (CE) from U937 cells (4 μg per sample) . Lanes 3 and 4 also contain 10 μM heparin (H) and 10 mM DPG (D) , respectively. Figure 2 is an autoradiogram from an in vi tro kinase assay that indicates PK-CKII and PKC induce IΔBα phosphorylation. ΔC (wt) I/cBc. was phosphorylated in vi tro with (A) purified PK-CKII (CKII) or (B) purified PKC (PKC) in the absence (lane 5) or presence of 10 μM heparin (H) (lane 6) or 10 itiM DPG (D) (lane 7) . Lanes 1 and 4 contain [ 35 S] -labelled ΔC IκBα to serve as a reference marker for migration of unphosphorylated IKBC.. Lane 2 contains ΔC I κBa substrate alone. Lane 3 contains the corresponding kinase alone. The lower panels are the corresponding Coomassie blue stained gels. [ 35 S] -labelled ΔC (wt) unphosphorylated I KBGI is marked with an arrow and the phosphorylated forms are marked with starred arrows . Figure 3 is an autoradiogram from an in vi tro kinase assay that indicates purified PK-CKII phosphorylates IKBC. at S32 and S36. ΔC (wt) IκBα (lanes 3 and 7) , ΔC 32/36 IKBCU mutant (32/36) (lanes 4 and 8) , ΔC 32 IKBCU mutant (32) (lane 5) or ΔC 36 IKBC. mutant (36) (lane 6) were phosphorylated in vi tro with purified PK- CKII (CKII) (lanes 3-6) or purified PKC (PKC) (lanes 7 and 8) . Lane 1 contains [ 35 S] -labelled ΔC (wt) I nBoi and lane 2 contains [ 35 S] -labelled ΔC 32/36 I/Bc. mutant. The lower panel contains the corresponding Coomassie blue stained gel. [ 35 S] -labelled unphosphorylated IκBa is marked with an arrow and the phosphorylated forms are marked with a starred arrow. Figure 4 is an autoradiogram from an in vi tro kinase assay that indicates cellular PK-CKII phosphorylates IKBC. at S32/S36. U937 total cell extract was immunoprecipitated (IP) with preimmune serum (PIS) (lane 2) or polyclonal antibodies to the c. and c.' catalytic subunits of PK-CKII (CKII) (lanes 3-6) followed by stringent washing and incubation with recombinant ΔC (wt) IKBC. (lanes 2, 3, 5 and 6) or ΔC 32/36 I κBa mutant (32/36) (lane 4). Lane 1 contains [ 35 S] -labelled ΔC (wt) I κBa . Lanes 2-4 are in the absence of 10 μM heparin. Lane 5 is in the presence of 10 μM heparin and lane 6 is in the presence of 10 mM DPG (D) . The lower panel contains the corresponding Coomassie blue stained gel . [ 35 S] -labelled ΔC (wt) unphosphorylated I/cBc. is marked with an arrow and the phosphorylated form is marked with a starred arrow. Figure 5 is an autoradiogram from an in vi tro kinase assay that indicates IKBO; interacts in vivo with a PK-CKII complex containing the a catalytic subunit. U937 total cell extract was immunoprecipitated (IP) with preimmune serum (PIS) (lane 1) , polyclonal anti-IκBα serum (A) (lane 2) , or polyclonal anti-IκB/3 serum (B) (lane 3) . The immunoprecipitated proteins (lanes 1-3) and 40 μg of U937 total cellular protein (lane 4) were run on a SDS polyacrylamide gel, transferred, and immunoblotted (IB) with anti-CKII antibody {a/a' ) (upper panel) , with anti-IκBα; antibody (middle panel) , and with anti-IκB3 antibody (bottom panel) . The arrows mark the corresponding proteins being immunoblotted. Figure 6 is an autoradiogram from an in vi tro kinase assay that indicates PK-CKII is not the I κBa S32/S36 kinase activity in the IKK Signalsome. Unstimulated HeLa extracts (lanes 1 and 2) , TNF stimulated (10 minutes at 10 ng/ml) HeLa extracts (lanes 3-10) or no extract (lane 11) were immunoprecipitated with preimmune serum (PIS) (lanes 1 and 3) or polyclonal anti-MKP-1 antibody followed by stringent washing and incubation with recombinant ΔC (wt) I κBa (lanes 1-4 and 6-11) or ΔC 32/36 IKBO; mutant (32/36) (lane 5) in an in vi tro kinase assay in the absence (lanes 1-5) or presence of 50 nM heparin (lane 6) , 100 nM heparin (lane 7) , 200 nM heparin (lane 8) , 1 πiM PK-CKII peptide inhibitor (lane 9) or 1 mM non-specific peptide inhibitor (lane 10) . ΔC (wt) IκBe. (lanes 12, 14-16), ΔC 32/36 I κ.Ba mutant (32/36) (lane 13) were phosphorylated in vi tro with purified PK- CKII (CKII) in the absence (lanes 12 and 13) or presence of 50 nM heparin (lane 14) , 1 mM PK-CKII peptide inhibitor (lane 15) or 1 mM non-specific inhibitor (lane 16) . Figure 7 is an autoradiogram from an in vi tro kinase assay that indicates I/cBα S32/S36 phosphorylation by PK-CKII is increased upon PMA stimulation or HIV infection. 100 μg of unstimulated (lane 1) , TNF stimulated (10 minutes at 10 ng/ml) (lane 2) , PMA stimulated (8 minutes at 50 ng/ml) (lane 3) or HIV infected (lane 4) U937 cell extract per sample was immunoprecipitated with polyclonal antibodies to the a and a' catalytic subunits of PK-CKII (CKII) followed by stringent washing and incubation with recombinant ΔC (wt) IKBC. in an in vi tro kinase assay. Detailed Description In one aspect, the invention features a method for identifying a compound that inhibits the phosphorylation of residues serine 32 (S32) and serine 36 (S36) of IKBQ; by CKII. The method includes incubating CKII and IcBc. polypeptide with the compound to determine if phosphorylation of residues serine 32 and serine 36 of IκBα is inhibited. As used herein, a "compound" refers to a biological macromolecule such as an oligonucleotide or a peptide, a chemical compound, a mixture of chemical compounds, or an extract isolated from bacterial, plant, fungal or animal matter. Protein Preparation CKII is a multifunctional serine/threonine protein kinase that is ubiquitously expressed and typically thought to be constitutively active in eukaryotic cells. CKII is highly conserved among eukaryotes, including yeast, Dictyostelium discoideum, C. elegans, Drosophila , plants, bovine and humans. In most species, CKII contains α and α ' catalytic subunits (37 to 44 kD) and a jβ subunit (24 to 28 kD) , and exists as an α α ' β 2 tetramer. The catalytic component can include an α/α' heterodimer, an a/ a homodimer, or an a ' I a' homodimer. The β subunit is thought to regulate the catalytic activity. Overall, CKII is thought to be minimally regulated, although its cellular function is poorly understood. CKII is located in the nucleus, cytoplasm and mitochondria of eukaryotic cells and also is known as phosvitin kinase, glycogen synthase 5 kinase, troponin T kinase and casein kinase G, based on its broad range of substrates. Unlike most kinases, CKII phosphorylates within acidic amino acid stretches like that at the C- terminus of IKBQ;. Serine residues 283, 288, 293 and threonine residue 291 of IKBQ; have been identified as sites located within the most C-terminal 40 amino acids of IKBC. that are constitutively phosphorylated in resting cells and may play a role in the constitutive or basal degradation of iKBα. McElhinney et al . , Mol . Cell. Biol. , 16:899-906 (1996). Allende et al . , FASEB J . , 9:313-323 (1995) . CKII is used herein interchangeably with "protein- kinase CKII" (PK-CKII) and refers to the holoenzyme, a catalytic subunit (s) , or a biologically active fragment of a catalytic subunit. Purified rat liver CKII can be obtained from Promega (Madison, I) . Alternatively, cell lysates can be used as a source of CKII. In general, cell lysates can be prepared by washing the cells in cold phosphate-buffered saline (PBS) and resuspending in a lysis buffer. Lysates can be prepared from cultured cells, HIV infected cells, or from isolated tissue such as liver. In general, cultured cells are prepared for lysis by washing in PBS prior to harvesting, then incubating with a lysis buffer in the cold. Cells also can be homogenized, for example, by briefly sonicating. Tissue samples are minced and then homogenized in the presence of a lysis buffer. Lysis buffers can include non-ionic detergents such as Igepal CA-630 and Triton X- 100 (Sigma Chemical Co., St. Louis, MO), chelating agents such as EDTA and EGTA, or high or low salt concentrations. Proteolytic activity within a lysate can be minimized by including protease inhibitors such as phenylmethylsulfonylfluoride (PMSF) , aprotinin, pepstatin and leupeptin. A typical lysis buffer can include, for example, 25 mM N-2-hydroxyethylpeperazine-N' -2- ethanesulfonic acid (HEPES) , pH 7.7, 0.3 M NaCl , 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM dithiothreitol (DTT), 20 mM jS-glycerophosphate, 0.1 mM Na 3 V0 4 , 2 μg/ml leupeptin and 100 μg/ml PMSF. After lysis of the cells, the lysate is centrifuged in the cold, i.e. 4°C, at 10,000-12,000 x g for about 15 minutes. CKII can be immunoprecipitated from a cell lysate with antibodies to the catalytic subunits. I κ.Ba polypeptide can be obtained through standard recombinant techniques. As used herein, " I κBa polypeptide" refers to an IKBO; polypeptide of any length that retains residues S32 and S36 in a configuration that permits phosphorylation of S32 and S36 by CKII. The nucleic acid sequence of I κBa has a GenBank accession number of M69043 gl87290. It should be noted that I/Bc. polypeptide containing one or more mutations is expected to serve as a substrate for CKII, provided that residues S32 and S36 are maintained. The cDNA encoding IκBc can be amplified by polymerase chain reaction (PCR) technology using oligonucleotides corresponding to the nucleic acid sequence of IκBa . Haskill, D. et al . , Cell , 65:1281-1289 (1991). PCR refers to a procedure or technique in which sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. The oligonucleotide primers are typically from about 15 to about 25 nucleotides in length. The sequence of the oligonucleotide primers can be modified. For example, restriction sites can be incorporated into the oligonucleotides to facilitate further manipulations of the DNA. A C-terminal truncation of full-length IκBa can be created by using oligonucleotides, for example, that incorporate a stop codon after amino acid 264. Mutations can be introduced in a similar fashion. A nucleic acid sequence encoding an IκBa polypeptide can be ligated into an expression vector and used to transform a bacterial or mammalian host cell. In bacterial systems, a strain of Escherichia coli such as BL-21 can be used. Suitable E. coli vectors include the pGEX series of vectors that produce fusion proteins with glutathione S-transferase (GST) . Transformed E. coli are typically grown exponentially, then stimulated with isopropylthiogalactopyranoside (IPTG) prior to harvesting. In general, such fusion proteins are soluble and can be purified easily from lysed cells by adsorption to gluathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety. In mammalian host cells, a number of viral-based expression systems can be utilized to express I/Bα polypeptides. A nucleic acid encoding an I κBα polypeptide can be cloned into a retroviral expression vector, such as that derived from the Spleen Focus Forming Virus (SFFV) . Fuhlbrigge, R.C. et al . , Proc . Natl. Acad. Sci . USA, 85 (15) : 5649-5653. The vectors can be introduced into a mammalian cell line by electroporation or other suitable method. Cell lines that are stably expressing I/.Be. polypeptides can be selected by a variety of selection systems, depending on the expression vector. For example, the neomycin gene confers resistance to the aminoglycoside G418. Alternatively, amplified sequences can be ligated into a mammalian expression vector such as pcDNA3 (Invitrogen, San Diego, CA) and then transcribed and translated in vitro using wheat germ extract or rabbit reticulocyte lysate . Phosphorylation Assays A phosphorylation or kinase assay is used to monitor the phosphorylation of residues S32 and S36 of I κBα polypeptide by CKII in the presence of a compound. An IκBe. polypeptide containing a C-terminal truncation of full-length IκBα is particularly useful in this assay. Appropriate assay conditions minimally include a buffer and a phosphate donor such as ATP or GTP . For example, a phosphorylation assay can include approximately 20 mM HEPES, pH 7.6, 20 mM MgCl 2 , 20 M 3-glycerophosphate, 20 mM p-nitrophenylphosphate (PNPP) , 0.1 mM Na 3 V0 4 , 2 mM DTT, 20 μM ATP and 0.1 μCi of [ 32 P] γ ATP. Alternatively, the phosphorylation assay can include 35 mM Tris HC1 , pH 7.5, 0.5 mM EGTA, 15 mM MgCl 2 , 1 mM NaCl , 0.1 mM CaCl 2 , 1 mM DTT, 60 mM ATP and 0.1 mCi of [ 32 P] γ ATP. Approximately 0.5 to 10 μg of IKBQ; polypeptide and about 2 units of purified CKII can be incubated in the presence of a compound. If a cell extract is the source of CKII, from about 50 μg to about 200 μg of total protein is used. Typically, from about 1 nM to about 1 mM of a compound can be included in the assay. After an initial screening, the optimal concentration of compound can be refined by using an entire range of concentrations. Phosphorylation assays are typically incubated from about 5 to about 50 minutes in length and are maintained at about 30°C. Preferably, the incubation is from about 30 to about 35 minutes. The length of incubation can be adapted to the incubation temperature. For example, if the incubation temperature is about 25°C, incubation time can be increased. Phosphorylation of residues S32 and S36 of I κBa polypeptide can be monitored by electrophoresing the protein mixture through a SDS polyacrylamide gel, which is then dried and exposed to x-ray film. The phosphorylation of IκBa polypeptide is compared with a corresponding assay in the absence of compound. Alternatively, inhibition of phosphorylation can be assessed by measuring the amount of radioactivity incorporated into I κBa polypeptide. In this method, the incubated mixture is placed onto cellulose filters (Whatman LabSales, Hillboro, OR) or other suitable material and then washed with 30% trichloroacetic acid (TCA) for 30 minutes at approximately 65°C. The cellulose filters are subsequently washed two to four times with 15% TCA for 15 minutes. After drying, the filters are washed in ethanol, dried and counted in a liquid scintillation counter. Casnellie, J.E., Meth. Enzvmol . , 200:155 (1991). Activation of NF/B-dependent genes also can be monitored as an assay of the phosphorylation of residues S32 and S36 of I κ.Ba . For example, an NF/cB luciferase reporter vector can be transfected into a cell line. Candidate compounds are added to the culture medium and cells are stimulated. Luciferase activity is then determined and compared with luciferase activity from stimulated cells incubated in the absence of compound. Mercurio, F., et al . , Science, 278:860-866 (1997). Other gene reporter systems known in the art can also be employed for monitoring activation of NFKB-dependent genes . CKII is a Moderately Inducible Kinase The invention also relates to a method to identify candidate compounds that selectively inhibits the up- regulation of CKII. The method includes contacting a cell preparation with a compound and monitoring the phosphorylation of residues S32 and S36 of I κBα polypeptide. An I KBOI polypeptide that is a C-terminal truncation of full-length IKBO; is useful in this method. A cell preparation can include viable cells or can be a cellular lysate. Cell preparations from HIV infected cells, malignant cells, and cells exposed to a tumor- promoting agent such as PMA are particularly useful . Surprisingly, CKII has been identified as a moderately inducible kinase. As shown herein, CKII can bind specifically to I κBα in vivo and phosphorylate residues S32 and S36 of IKBC.. This stimulus-induced hyperphosphorylation event is characterized by its ability to cause IκBα to migrate slower on an SDS polyacrylamide gel and leads to I κBα degradation in vivo . Moderate or chronic activators of NF/cB, including HIV and PMA, can cause an increase in CKII specific I κBα S32/S36 kinase activity. TNF, a rapid and strong inducer of NFKB, does not appear to cause an increase in CKII specific IKBQ; S32/S36 kinase activity. Persistence of HIV infection is associated with an increase of nuclear NFcB levels, which favor viral replication. The increase of nuclear NFcB is thought to be a result of selective degradation of I/cBc.. Deletion of NF-KB bindings sites within the HIV promoter abrogates HIV replication. It has previously been shown that mutation of S32 and S36 each to alanine abrogates the formation of the slower migrating I nBot and de novo phosphorylation of I κBα had been observed in the N-terminal region containing these two sites. This has led to the hypothesis that serines 32 and 36 are targets of an inducible I KBOI kinase. S32 and S36 are required for TNF, PMA-ionomycin and LPS induced I κBa degradation. Several kinases have been identified that target the N-terminal region of I/cBof, which is not surprising as there are well over one hundred different stimuli that can activate NF/cB. A tyrosine kinase activity elicited by treatment of cells with pervanadate was demonstrated to phosphorylate Y42, but not S32 or S36, causing the slower migration of I κBa on a polyacrylamide gel and activation of NF-/cB. Imbert et al . , Cell, 86:787-798 (1996) . This activation appears to be a unique proteolysis-independent mechanism of NF/cB activation. Ribosomal S6 kinase (p90 rskl ) can phosphorylate S32 and appears to be necessary but not sufficient for mitogen induced NF/cB activation. As shown herein, protein Kinase C phosphorylates I/cBc. at amino acids other than S32/S36. It is noteworthy to point out that such PKC-dependent phosphorylation results in a characteristic slower migration of IcBc. which was thought to result from the phosphorylation of S32/S36 or as recently shown, phosphorylation of Y42. In addition, a large (700 kD) multisubunit complex purified from unstimulated cellular extracts was shown to contain an I/cBc. kinase activity that targeted S32/S36 in the presence of ubiquitin and ubiquitin enzymes. Chen et al . , Cell, 84:853-862 (1996). Several TNF inducible components of this high molecular weight complex (IKK Signalsome) have been identified. Mercurio et al . , Science, 278:860-866 (1997); and DiDonato et al . , Nature , 388:548-554 (1997). IKKc. (CHUK, IKK-1) and IKK/3 (IKK-2) are kinases within this complex which, when immunoprecipitated from in vi tro translation systems or from cell lines, can phosphorylate I/cBc.. While S32 can be phosphorylated by either IKK kinase, S36 is a better target for IKKo;. It appears that both IKKo; and IKK3 are important regulators of certain inducible NF/cB activation pathways, although there are some inconsistencies between studies involving anti-sense and kinase dead IKKα;//3 inhibition of NF/cB activation. It is still unknown, however, whether pure IKKct; and/or IKK/3 can directly phosphorylate I κBa at S32 and/or S36. As described herein, HIV infection up-regulates the activity of protein kinase CKII but does not regulate activity of two of the other kinases that phosphorylate I/cBcx at S32/36: IKK complex and pp 90rskl . These results imply that HIV infection selectively regulates CKII activity, which in turn targets the phosphorylation of S32/36 to induce I κBa degradation. The identification of compounds that selectively interfere with CKII up- regulation by HIV, or more importantly and specifically, with the phosphorylation of I/cBcx on serine 32/36 by CKII would potentially provide medically significant compounds that influence the activation of NF/cB by HIV infection and ultimately affect viral persistence. When considering an increase in the phosphorylation level of a substrate, two different enzymatic activities need to be included: phosphorylation and dephosphorylation. In unstimulated U937 cells, constitutive in vivo PK-CKII specific phosphorylation of I/cBcϋ at S32/S36 may be visually masked by a powerful phosphatase activity at these sites. Upon cellular stimulation by HIV, PMA or another moderate stimulus, this delicate kinase-phosphatase balance may be disrupted due to a loss of phosphatase activity, allowing I/cBo; to accumulate in the S32/S36 phosphorylated "upshifted" molecular form. Use of purified PK-CKII, immunoprecipitated PK-CKII that was stringently washed or the presence of potent phosphatase inhibitors such as okadaic acid in the kinase assays eliminated any phosphatase activity, thus potentially favoring the effects of PK-CKII activity. Knowing that purified PK- CKII (without any associated phosphatase effects) can phosphorylate I/cBc. at S32/S36 causing the characteristic protein upshift previously linked to I/cBo; degradation, suggests a potential "PK-CKII -phosphatase balance" model for I/cBo; control. Importantly, with the identification of PK-CKII as a S32/S36 I/cBc. kinase, it is now possible to study the effects of certain purified phosphatases using the models described herein. Another possible explanation for the observed S32/S36 phosphorylation only after moderate stimulation with HIV (or PMA, etc.) may involve the accessibility of these sites to PK-CKII. In non-activated cells, S32/S36 may be physically masked by another IcBc. domain or by an unrelated associated protein { e . g. p65) . Cellular stimulation may lead to a conformational change in 1/cBcκ or of an associated protein which results in the accessibility of S32/S36 to a constitutively active PK- CKII. For example, it can be hypothesized that cell activation results in phosphorylation or dephosphorylation at other target I κBa amino acids (such as the C-terminal PK-CKII sites) or of associated proteins, thus exposing S32/S36 to PK-CKII. In this system, in order to eliminate the massive PK-CKII phosphorylation of I κBa at its C-terminus which would potentially overwhelm any additional phosphorylation event, an I/cBα; construct that is truncated after amino acid 264 can be used. By doing this, the domain responsible for blocking the S32/S36 sites may be eliminated. In the experiments reported below, full length I/cBcv that was transcribed/translated in vi tro using a wheat germ extract system appeared to be upshifted by immunoprecipitated PK-CKII, at least to a modest extent. PK-CKII may be regulated in vivo by upstream regulatory proteins that influence its activity as an S32/S36 I κBa kinase. A recent report indicates that MEKKl is upstream and regulates the putative 700 kD multisubunit I/cBc kinase activity containing complex. Lee et al . , Cell, 88:213-222 (1997) Since PK-CKII can phosphorylate I κBa at S32/S36, it is a prime target in the control of I κBa degradation and consequently, NF-/cB activation. The identification of a specific physical interaction between the PK-CKII a catalytic subunit and not the c.' subunit with I/cBα aids in the potential targeting of PK-CKII. Disruption of both catalytic subunits of PK-CKII in S. cerevisiae results in lethality, although disruption of the α subunit alone presents a wild-type phenotype. Padmanabha et al., Mol. Cell. Biol . , 10:4089-4099 (1990); Chen-Wu et al., Mol. Cell. Biol. , 8:4981-4990 (1988). If the α subunit of PK-CKII is effective for binding lκBα , but α' alone can orchestrate the other normal PK-CKII dependent functioning of a cell, disruption or down-regulation of the ex subunit of PK-CKII alone could potentially safeguard against stimulus-induced NFkB activation without loss of cell viability. The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. Examples Example 1 - Reagents and Methods for Phosphorylation Assays : Plasmids and recombinant I κBα - The I/cBo; (MAD3) cDNA plasmid was obtained from Cetus Corporation and used as a template for subsequent PCR amplification. Haskill, D. et al . , Cell, 65:1281-1289 (1991) . The full-length I/Bα MAD3 sequence (amino acids 1-317) was PCR amplified and ligated into pGEX-KG (derived from pGEX-2T from Pharmacia Biotech, Piscataway, NJ) . McElhinney et al . , Mol. Cell. Biol., 16:899-906 (1996) . An I/cBα mutant lacking the C-terminal portion (ΔC) was amplified in a manner similar to the wt , using a 3' antisense primer that created a stop after amino acid 264. A double S32A/S36A mutant was amplified off the ΔC I/cBc. species using PCR primers that created alanines at amino acids 32 and 36 and was named ΔC 32/36. Full length constructs of I/cBo; with single mutations at S32 or S36 (alanines) were obtained from Dr. Dean Ballard of Vanderbilt University (Nashville, TN) and were used to create ΔC 32 and ΔC 36, as described above. All were ligated into pGEX-KG and transformed into Escherichia coli BL-21 cells. These were grown exponentially, and after one hour of stimulation with isopropylthiogalactopyranoside (IPTG) , each glutathione- S-transferase (GST) construct was isolated. The purity of the proteins was analyzed by SDS-PAGE and considered to be greater than 90%. The GST portion of the protein was then removed by thrombin cleavage using 10 ng thrombin (Sigma, St. Louis, MO) per microgram of protein in thrombin cleavage buffer (50 mM Tris-HCl ph 7.5 , 2.5 mM CaCl 2 , 50 mM NaCl) . After a 10 minute incubation at 25 °C, 1 μg/μl PMSF was added and complete cleavage was confirmed by SDS-PAGE. The ΔC constructs were also ligated into the pcDNA3 mammalian expression vector (Invitrogen, San Diego, CA) and transcribed/translated in vi tro using the TNT Coupled Wheat Germ System (Promega, Madison, WI) according to the manufacturer's instructions . Reagents - Heparin (H-3149) was obtained from Sigma Chemical Co. (St. Louis, MO) and solutions were made using an average molecular weight of 16,000. 2,3- diphospho-D-glyceric acid (DPG) was also obtained from Sigma Chemical Co. (St. Louis, MO). Purified rat liver PK-CKII was obtained from Promega (Madison, WI) and purified PKC (mixture of a, β , and γ) was obtained from Upstate Biotechnology Incorporated (UBI , Lake Placid, NY) . PMA was obtained from Sigma Chemical Co. (St. Louis, MO) and TNF was purchased from Genzyme (Cambridge, MA) . Anti-human PK-CKII (polyclonal IgGl) was also obtained from UBI and antibodies to the catalytic subunits of PK-CKII {a and a' ) were provided by Dr. Daniel R. Marshak of Osiris Therapeutics, Inc., Baltimore, MD . Chester et al . , J. Biol . Chem. , 270:7501- 7514 (1995) . Anti-MKP-1 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and a rabbit polyclonal anti-I/cBα (MAD3) serum was generated as described by McElhinney et al . , J. Virol. , 69:1500-1509 (1995). A polyclonal anti-I/cBα (MAD3) antibody raised against amino acids 6-20 (sc-203) and its control peptide (sc-203 P) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) . Anti-I/B/3 antibodies were generated from sera of rabbits immunized with recombinant murine IcB/3. The cDNA of I/cB/3 was provided by Dr. Sankar Ghosh of Yale University, New Haven, CT. Cell culture, HIV infection, cellular stimulation and protein extraction - U937 pro-monocytic cells were obtained from the American Type Culture Collection (ATCC accession number CRL 1593) and grown in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 5% heat-inactivated fetal bovine serum (FBS, Intergen, Purchase, NY) , 100 U of penicillin/streptomycin per ml and 2 mM L-glutamine and grown to a density of 4 x 10 s to 6 x 10 5 cells per ml. HeLa cells were obtained from the ATCC (accession number CCL2) and grown in DMEM (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated FBS, 100 U of penicillin/streptomycin per ml, and 2 mM L-glutamine. U937 cells were stimulated for 8 minutes with 50 ng/ml PMA or infected with HIV-1 LAV-Bru strain and studied at days 20-90 post-infection. McElhinny, J.A. et al . , J. Virol. , 69:1500-1509 1995. U937 and HeLa cells were stimulated for 10 minutes with 10 ng/ml TNF. U937 cellular lysates were obtained by washing cells with cold PBS and resuspending in whole cell extract (WCE) buffer containing 25 mM HEPES, pH 7.7, 0.3 M NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM DTT, 20 mM /3-glycerophosphate, 0.1 mM Na 3 V0 4 , 2 μg/ml leupeptin and 100 μg/ml PMSF. After incubation on ice for about 5 to about 10 minutes, lysates were centrifuged at 12,000 x g for 15 minutes. HeLa cellular extracts were obtained by washing cells with cold PBS, resuspending in PD-500 buffer containing 40 mM Tris HC1 , pH 8, 50 mM NaCl , 0.1% NP40, 6 mM EDTA, 6 mM EGTA, 10 mM /3-glycerophosphate, 10 mM NaF, 10 mM p- nitrophenylphosphate (PNPP) , 300 mM Na 3 V0 4 , 1 mM DTT, 1 mg/ml leupeptin, 10 mg/ml aprotinin, 1 mg/ml pepstatin and 2 mM PMSF. After incubation on ice for about 5 to about 10 minutes, the lysates were centrifuged at 12,000 x g for 15 minutes. Protein content of the clarified supernatant was calculated using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA) . Phosphorylation assays - Recombinant I κBa (2-6 μg, with or without the GST moiety) was incubated with or without an enzyme source in Kinase Buffer A (20 mM HEPES pH 7.6, 20 mM MgCl 2 , 20 mM /3-glycerophosphate, 20 mM PNPP, 0.1 mM Na 3 V0 4 , 2 mM DTT) with 20 μM ATP and 0.1 μCi of [γ- 32 P ] ATp for 3Q m i nu tes at 30°C or in Kinase Buffer B (35 mM Tris HC1 , pH 7.5, 0.5 mM EGTA, 15 mM MgCl 2 , 1 mM NaCl, 0.1 mM CaCl 2 , 1 mM DTT) with 60 mM ATP and 0.1 mCi of [ 32 P] γ ATP for 35 minutes at 30°C. Samples were then resolved by SDS-PAGE, dried and visualized by autoradiography or transferred to an Immobilon-P membrane (Millipore, Bedford, MA) and visualized by autoradiography. To assess protein content, the gels were rehydrated and Coomassie stained. For assays using PKC as the kinase source, Kinase Buffer A was used with the addition of 0.1 mM CaCl 2 . Immunoprecipitations and Western Blotting - When immunoprecipitating PK-CKII or the IKK Signalsome to be used in a kinase assay, 100-200 μg of cellular extract was incubated with pre-immune sera, antibodies to the catalytic subunits of PK-CKII {a and a ' ) or antibodies against MKP-1 and Protein A agarose (Gibco BRL, Grand Island, NY) for 90 minutes at 4°C. The beads were then stringently washed with Wash Buffer #1 containing 20 mM Tris base, 40 mM NaCl, 5 mM EDTA, 50 mM NaF, 30 mM Na 4 P 2 0 7 , 1% Triton X-100, 0.1% SDS and 1% deoxycholate and used in the kinase assay. To determine protein associations, 500 μg of cellular extract was incubated with the appropriate antibody for 90 minutes at 4°C. Protein A agarose was then added and incubated for one hour at 4°C. The beads were washed in 50 mM Tris-HCl pH 7.5 , 50 mM NaCl, 1% Triton X-100, 2 mM EDTA, 20 mM NaF and 100 mM β glycerophosphate and the protein was eluted. Eluants were separated by SDS-PAGE and transferred to an Immobilon-P membrane by standard procedures. Immunoblotting was done with specific antibodies and visualized using the ECL Western blotting detection kit (Amersham, Buckinghamshire, England) . Example 2 - PK-CKII participates in the induction of a slower migrating molecular form of I/cBα: In order to test whether PK-CKII phosphorylates I κBa at S32/S36, an in vi tro phosphorylation assay was developed. Full length wild type (wt) IκBa was transcribed and translated in wheat germ extract in the presence of [ 35 S] -methionine followed by immunoprecipitation with anti-I/cBc. peptide antibodies and dissociation of the [ 35 S] -labelled I/cBc from the antibody by incubation with an excess of the corresponding I κBa peptide. [ 35 S] -labelled products were assessed by SDS-PAGE and the [ 35 S] -labelled IcBc. molecules were then isolated from the wheat germ extract by immunoprecipitation with an anti-IcBc. antibody directed against amino acids 6-20 (Santa Cruz, see below) . Following stringent washing, the I/cBcx molecules were dissociated from antibody using the control peptide for the antibody (amino acids 6-20) . This process resulted in a recombinant I κBa molecule void of material that was present in the wheat germ extract which could potentially enhance or inhibit the phosphorylation or dephosphorylation of I/cBc. by putative kinases or phosphatases . Phosphorylation assays with [ 35 S] -labelled substrates were performed by incubation with or without an enzyme source in kinase buffer (20 mM Tris-HCl, 0.5 mM DTT, 10 mM MgCl 2 , 2 mM ATP, 0.5 μM okadaic acid) for one hour at 30°C. Samples were then resolved by SDS-PAGE, dried, and visualized by autoradiography. As shown in Fig. 1, this resulted in the appearance of a slower migrating form of [ 35 S] -labelled I/cBo; (lane 2) . Incubation with cell extracts in the presence of known CKII inhibitors (10 μM heparin or 10 mM 2,3 DPG) resulted in the complete loss of this slower migrating form of I/cBo; (lanes 3 and 4, respectively) . These results suggest that PK-CKII is present in the cell extracts and is involved in the post-translational modification of I/cBc. that causes its slower migration on the gel . Example 3 - PK-CKII and PKC phosphorylate ΔC IκBc: To verify that the upshift of I/cBα observed in Figure 1 was due to phosphorylation and not to another post- translational modification influencing the mobility of I/cBcx on a SDS polyacrylamide gel, incorporation of [ 32 P]γ- ATP into I/cBc. was analyzed. Due to the known PK-CKII multi-site phosphorylation at the C-terminal end of I κBa , a I κBa substrate lacking the C-terminus (ΔC) was used. In vi tro kinase assays were performed with Kinase Buffer A as described in Example 1. Addition of purified PK-CKII to a ΔC wild type (wt) I/cBc. polypeptide in the presence of [ 32 P] -γ-ATP induced the de novo phosphorylation of a protein (Figure 2A, lane 5) which in its phosphorylated form, migrates slower than the [ 35 S] -labelled ΔC I/cBc. incubated in the absence of PK-CKII (lane 4) . When the ΔC (wt) I/cBo; is incubated with purified PK-CKII in the presence of heparin or 2 , 3 DPG, [ 32 P] incorporation was not observed (lane 6 and 7, respectively), confirming the role of PK- CKII in this phosphorylation event. Additional controls in this experiment included the separate incubation of ΔC (wt) I κBa (lane 2) or purified PK-CKII (lane 3) in the in vi tro kinase assay with [ 32 P] -γ-ATP, which resulted in no detectable phosphorylation. Equal loading of IκBa in each reaction was verified by staining the membrane with Coomassie blue. Of interest, the slower migrating [ 32 P] - labelled ΔC (wt) I/cBo. was not observed by Coomassie staining suggesting that only a small fraction of the substrate is phosphorylated and hence being converted into a slower migrating form. PKC, an enzyme previously suggested to directly phosphorylate I κBa, was tested to determine if it induces the phosphorylation of I/cBα with the same characteristics as those observed for PK-CKII in Figure 2A. Using the same experimental design, ΔC (wt) I κBa was incubated in the presence or absence of purified PKC {a , β , and γ) in an in vi tro kinase assay as described in Example 1. As shown in Figure 2B, this results in the appearance of two separate [ 32 P] -labelled bands (lane 5) . The faster migrating [ 32 P] -labelled protein migrates at the same level as the migration marker, [ 35 S] -labelled ΔC (wt) I/cBα (lane 4) , while the slower migrating [ 3 P] -labelled protein migrates at a similar (yet perhaps distinct) level as the one observed for the ΔC (wt) I/cBc phosphorylated by purified PK-CKII (compare to Figure 2A, lane 5) . Importantly, addition of heparin or 2 , 3 DPG did not alter significantly the PKC-induced phosphorylation of the ΔC (wt) I/cBo; (Figure 2B, lanes 6 and 7) . Autophosphorylation of the classical PKC isoenzymes {a , β , and γ) when PKC is incubated in the in vi tro kinase assay with [ 32 P] γ-ATP (Figure 2B, lanes 3, 5-7) is also observed. As before, the SDS-PAGE gel was rehydrated and stained with Coomassie blue to demonstrate equal loading of ΔC (wt) I/cBo; in each reaction. Example 4 - PK-CKII but not PKC (at, β, and γ) phosphorylates lκBa at S32 and/or S36: Knowing that the ΔC (wt) I/cBc. substrate includes multiple amino acids that could be targets of either PK-CKII or PKC {a , β , and 7) , experiments were performed to test whether purified PK- CKII can phosphorylate I/cBα at residues S32 and S36. For this, residues S32 and S36 of the ΔC I/cBc substrate were mutated to alanine (ΔC 32/36) as described in Example 1. The ΔC (wt) I/cBc. or the ΔC 32/36 I/cBc. mutant were separately incubated with purified PK-CKII in an in vi tro kinase assay containing [ 32 P] γ-ATP. As shown in Figure 3, the same [ 32 P] -labelled slower migrating ΔC (wt) I/cBc previously detected (Figure 2A, lane 5) was again observed (Figure 3, lane 3). However, this [ 32 P] -labelled protein was not present when the ΔC 32/36 I/cBα mutant was incubated with purified PK-CKII (Figure 3, lane 4) . The sequences flanking both S32 and S36 make them each a classic consensus PK-CKII site. Because there are controversial reports as to whether each serine individually is targeted by a putative N-terminus I/cBα kinase, it was determined whether purified PK-CKII requires the presence of both serines to phosphorylate I/cBα and cause its slower migration. For this, I/cBc. substrates were used in which each serine was individually mutated to alanine yielding a ΔC 32 I/cBα and a ΔC 36 I/cBc. as described in Example 1. These two I/cBα mutants were then separately incubated with purified PK- CKII in an in vi tro kinase assay containing [ 32 P] γ-ATP. As shown in Figure 3, mutation of either S32 (lane 5) or S36 (lane 6) alone did not abrogate the phosphorylation of ΔC I/cBc. or its slower migration resulting in a similar pattern as that observed with the ΔC (wt) I κBa (lane 3) . Altogether, the results indicated that purified PK-CKII can phosphorylate S32 or S36 individually causing the characteristic change in protein mobility. Having shown that PKC can directly phosphorylate I/cBc. and surprisingly, cause the appearance of a slower migrating form, it was determined whether such phosphorylation events target S32 and S36. For this, ΔC (wt) I/cBα or ΔC 32/36 IcBcv mutants were incubated with purified PKC {a , β , and γ) and analyzed for [ 32 P] incorporation and migration characteristics. As shown in Figure 3, PKC induced the same two [ 32 P] -labelled forms of I/cBo; previously shown in Figure 2B (lane 5) regardless of mutation of S32/S36 (Figure 3, compare lanes 7 and 8) . This suggested that PKC targets amino acids other than S32/S36 which unexpectedly also induce a change in the migration of I/cBc. As with previous experiments, equal loading of I/cBo; constructs in each reaction was verified by Coomassie blue staining. Example 5 - Native PK-CKII phosphorylates IKBC. at S32/S36: Having demonstrated that purified PK-CKII phosphorylates I κBa at S32 and S36, the ability of native PK-CKII, isolated from U937 cells, to function as a S32/S36 I/cBα kinase was assessed. The a and a' catalytic subunits of PK-CKII were co-immunoprecipitated from 200 μg of cell lysate using specific antipeptide (PK-CKII a and a' ) polyclonal serum and washed in high salt buffer to eliminate any associated proteins. Immunoprecipitates were then incubated with either ΔC (wt) IκBa or ΔC 32/36 I/cBo; mutant in an in vi tro kinase assay containing Kinase Buffer A and [ 32 P] γ-ATP. As shown in Figure 4, immunoprecipitated PK-CKII induced the phosphorylation and slower migration of the ΔC (wt) I/cBo; (lane 3) but not of the ΔC 32/32 I/cBc mutant. As a control, immunoprecipitates of cell lysates using preimmune sera did not induce the phosphorylation and slower migration of ΔC (wt) I/cBα, although a small degree of phosphorylation was observed (lane 2) in a protein migrating the same level as the unphosphorylated [ 35 S] - labelled ΔC (wt) I/cBc. (lane 1) . As expected, heparin or 2,3 DPG inhibited the immunoprecipitated PK-CKII -induced phosphorylation of the ΔC (wt) I/cBo;. Equal loading of the ΔC I/cBo; constructs was verified by Coomassie blue staining of the gel. In addition, immunoprecipitation of PK-CKII was verified by analyzing parallel immunoprecipitates from cell lysates by SDS-PAGE, followed by immunoblotting with anti-PK-CKII antibodies. Example 6 - In vivo interaction between I/cBα and a PK-CKII complex that inhibits the catalytic subunit: PK-CKII exists in cells as a heterotetramer containing two catalytic and two regulatory subunits. The catalytic component consists of an oj/c' heterodimer, an cϋ/α; homodimer, or an α'/α' homodimer. Demonstrating an in vivo interaction between I κ.Ba and a catalytic subunit (s) of PK-CKII would help to identify which catalytic subunit (s) are responsible for the I/cBc. phosphorylation at S32/S36. To determine whether and which PK-CKII catalytic subunit (s) associates with I/cBc, 500 μg of cell lysates were immunoprecipitated with several antibodies including anti-I/cBo;. The immunoprecipitates were then analyzed by SDS-PAGE and immunoblotted with anti-PK-CKII a and a ' antibodies. As shown in Figure 5, neither preimmune sera nor anti-IcB/3 antibodies co- immunoprecipitated either catalytic subunit of PK-CKII (lanes 1 and 3) . On the contrary, anti-I/cBo; antibodies co- immunoprecipitated a protein that is immunoreactive with the c subunit of PK-CKII (lane 2) . Each immunoprecipitation was subsequently immunoblotted with the appropriate antibody to demonstrate the precipitation of the corresponding molecule. These results lend evidence to the specificity of the in vivo PK-CKII-1 /cBe. interaction and demonstrate that the PK-CKIIo; catalytic subunit is a potential target for further in vivo functional analysis. Example 7 - PK-CKII does not appear to be the I/cBc S32/36 activity in the IKK signalsome: A high molecular mass multiprotein complex that contains an inducible I κBa (S32/36) kinase activity has been identified. This I/cB kinase (IKK) signalsome contains at least two kinase activities, IKKc (CHUK, IKK-1) and IKK/3 (IKK-2) , that are hypothesized to be direct I/cBα kinases. However, no pure kinase found within this IKK signalsome has yet been shown to directly phosphorylate I/cBα at S32 and/or S36. To determine whether PK-CKII can be isolated from the IKK signalsome, the IKK signalsome was isolated from HeLa cells by immunoprecipitation using a specific anti-MKP-1 antibody. Mercurio, F. et al . , Science, 278:860-866 (1997) . Unstimulated and TNF stimulated HeLa cells were lysed, immunoprecipitated with the specific anti-MKP-1 antibody or preimmune sera, and washed with high salt buffer. Immunoprecipitates were then incubated with ΔC (wt) IκBe. in an in vi tro kinase assay containing [ 32 P] γ- ATP. As expected, the I/cBc kinase activity of the immunoprecipitated IKK Signalsome was induced upon TNF stimulation (Figure 6, lanes 2 and 4) while there was no detectable phosphorylation in the preimmune sera immunoprecipitation (lanes 1 and 3) . Use of the ΔC 32/36 substrate resulted in no detectable I κBa phosphorylation (lane 5) . In order to determine whether PK-CKII is responsible for the I/cBα S32/36 phosphorylation in the IKK signalsome, two different inhibitors of PK-CKII were utilized. Neither increasing concentrations of heparin (lanes 6-8) nor a specific PK-CKII pseudosubstrate peptide inhibitor (lane 9) affected the ability of the signalsome to inducibly phosphorylate ΔC I/cBc.. Both heparin (lane 14) and the peptide inhibitor (lane 15), but not the non-specific peptide (lane 16) , could inhibit the 32/36 specific phosphorylation of ΔC I/cBo; by pure PK- CKII (lanes 12 and 13) . Disregarding potential problems of a kinase within the signalsome being inaccessible to specific inhibitors, this suggests that PK-CKII is not the enzyme responsible for the inducible I Bα S32/36 kinase activity within the IKK signalsome. Example 8 - IcBα S32/36 specific phosphorylation by PK-CKII is increased upon PMA stimulation or HIV infection: Stimuli other than TNF that induce NF/cB activation have been shown to also target S32 and S36 of I κBa . Since PK-CKII is not a highly inducible kinase, stimuli that induce a less rapid and robust activation of NF/cB were tested. Unstimulated, TNF stimulated, PMA stimulated and HIV infected U937 cells were lysed, immunoprecipitated with anti-CKII a and a' , and subjected to high salt washing. Immunoprecipitates were then incubated with ΔC (wt) I/cBc. in an in vi tro kinase assay containing Kinase Buffer B and [ 32 P] γ-ATP. While TNF was unable to up-regulate PK-CKII specific I/cBc. S32/36 phosphorylation (Figure 7, lane 2), both PMA (lane 3) and HIV infection (lane 4) significantly induced PK-CKII specific phosphorylation at these sites. This suggests a putative mechanism by which PMA and HIV target I κBa for degradation. Example 9 - HIV infection of Monocytic Cell Lines Targets the Phosphorylation of IκBα at S32/S36: Mutations were generated by site-directed mutagenesis using PCR-based techniques to replace serine, threonine, or tyrosine residues by alanines (phenyl-alanine in the case of tyrosine) . A number of mutations were introduced that included serine 32/36, CKII sites, tyrosine 46, PKC sites, and combinations thereof. A FLAG epitope sequence was added to aid in subsequent purification. These I κBa transgenes were cloned into a retroviral expression vector (SFFV) , followed by their electroporation into the U937 monocytic cell line. Cells that stably expressed the I κBa tagged transgene were confirmed by their ability to grow in the presence of G418. These cultures were immediately subcloned to generate a number of clones, which were then verified for the expression of the I/cBo; tagged gene by immunoblotting using anti-FLAG antibodies. Using this approach, a number of clones were generated containing each of the different I κBa mutants and subclassified according to their level of expression of the transgene . Each pool of U937 clones expressing the appropriate I κBa transgene was compared to a pool of clones of U937 that contained the SFFV vector alone. The cells were infected with HIV as described in Example 1. After chronic infection had been established (normally by 20 days post-infection) , NF/cB levels in the nuclei of HIV-infected cells were compared with that of uninfected cells through gel-shift experiments. HIV infection of transfected U937 cells induced significant NF/cB activity (p50 and p65) as determined by the supershifting. U937 cells, however, that overexpressed the I/cBc. S32/36 mutant transgenes, but not the other mutations, were refractory to NFcB induction by HIV. Other Embodiments It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims: Claims What is claimed is: 1. A method to identify a compound inhibiting the phosphorylation of residues serine 32 and serine 36 of I/cBc. comprising incubating an I/cBc. polypeptide and CKII with said compound and determining if phosphorylation of said residues is inhibited. 2. The method of claim 1, wherein said I/cBα polypeptide is a C-terminal truncation of full-length 3. The method of claim 1, wherein said CKII is from an HIV infected cell. 4. The method of claim 1, wherein said CKII consists essentially of the alpha and alpha' catalytic subunits of the CKII holoenzyme. 5. The method of claim 1, wherein said CKII consists essentially of the alpha subunit of the CKII holoenzyme . 6. A method to identify candidate compounds selectively inhibiting the up-regulation of CKII comprising contacting a cell preparation with a compound and monitoring the phosphorylation of residues serine 32 and serine 36 of I/cBα polypeptide. 7. The method of claim 4, wherein said I/cBc. polypeptide is a C-terminal truncation of full-length I κBa . 8. The method of claim 6, wherein said CKII consists essentially of the alpha and alpha' catalytic subunits of the CKII holoenzyme. 9. The method of claim 6, wherein said CKII consists essentially of the alpha subunit of the CKII holoenzyme . 10. The method of claim 6, wherein said cell preparation comprises viable cells. 11. The method of claim 6, wherein said cell preparation comprises a cellular lysate. 12. The method of claim 6, wherein said cell preparation is from HIV infected cells. 13. The method of claim 6, wherein said cell preparation is from malignant cells. 14. The method of claim 6, wherein said cell preparation is from cells exposed to a tumor promoter. 15. The method of claim 14, wherein said tumor promoter is PMA.

Inventors: BREN GARY D, PAYA CARLOS V, TAYLOR JULIE A, BREN, GARY, D., PAYA, CARLOS, V., TAYLOR, JULIE, A.
Applicants: MAYO FOUNDATION, MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH
Application year: 1998
Main classification: G01N33
IPC codes: C12Q1, G01N33