Phosphorylation of IκBβ is required to mediate the dissociation of NFκB from DNA Alexandra Krstic, Holly E. Dembinski, Kevin Wismer, and Elizabeth Komives, PhD

University of California, San Diego • Department of Chemistry and Biochemistry • 9500 Gilman Drive, La Jolla, California 92093-0378

Nuclear factor κB (NFκB) is responsible for the regulation of genes implicated in tumor suppression, inflammatory response, development, and apoptosis. When NFκB fails to regulate gene expression, diseases such as cancer, chronic inflammation, and Alzheimer’s result. NFκB is regulated by its inhibitor, IκB, one of which is IκBβ. Like its more prominent counterpart IκBα, IκBβ prevents gene expression by remaining bound to NFκB in the cytoplasm and masking its nuclear localization sequence (NLS), stopping NFκB from entering the nucleus. Unlike IκBα, however, not all IκBβ immediately masks the NLS when associating with NFκB. Thus, some IκBβ remains bound to NFκB in the nucleus, inhibiting IκBα’s ability to bind to NFκB and prolonging gene expression. In this way, IκBβ both inhibits and activates gene expression.1 Until recently, the mechanism by which IκBβ fulfils both these functions were unclear. In vivo work in the Sankar Ghosh lab at Columbia University has shown that phosphorylated IκBβ can regulate NFκB binding to DNA; however, hypophosphorylated IκBβ forms an NFκB:DNA:IκBβ complex insusceptible to regulation by IκBα. Here we present progress towards confirming that phosphorylation of IκBβ is required for IκBβ-mediated dissociation of NFκB from DNA. We have restored a mutated plasmid to express wild type IκBβ and introduced a phosphomimic mutation (S346D) via polymerase chain reaction (PCR). We have also recombinantly expressed and purified Mus musculus IκBβ and p65 NFκB and have laid the groundwork for kinetic assays to investigate the significance of the phosphorylation state of IκBβ in NFκB regulation.

References 1. S. Ghosh et al. IκBβ acts to both inhibit and activate gene expression at different stages in the inflammatory response Nature

(2010). 2. K. Tran, D.T. Distinct Functional Properties of IκBα and IκBβ Molecular and Cellular Biology (1997).

Future Directions Having purified nearly all the necessary proteins (save the S346D mutant IκBβ), we plan to begin kinetic assays using stopped flow fluorescence to measure the relative rates of wild type IκBβ-mediated dissociation and of S346 mutant IκBβ-mediated dissociation of NFκB from DNA. This will allow us to determine whether phosphorylation of IκBβ is crucial to IκBβ’s functionality.

Acknowledgements Sincerest thanks to Professor Komives for welcoming me into her lab; to Holly Dembinski for being a truly incredible mentor; and to Kevin Wismer for teaching/putting up with me in the lab. This work was funded in part by NIH P01-GM071862-07.

IκBs regulate NFκB

IκBβ is a six ankyrin repeat protein with a C-terminal PEST sequence and an intrinsically disordered linker region between AR3 and AR4. The IκBβ PEST sequence gets phosphorylated in vivo by a casein kinase II-like protein, thus increasing the negative charge on the PEST sequence.2 We hypothesize that the negatively charged PEST sequence is crucial to IκBβ-mediated dissociation of NFκB from DNA by eletrostatically repelling DNA from the IκBβ:NFκB complex. By contrast, hypophosphorylated IκBβ is unable mediate dissociation. We plan to test this by introducing a phosphomimic mutation (S346).

Repairing plasmid

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3 126 G G H T A L H L A C R V R A H T C A C V L L Q P R P S H P R 156 D A S D T Y L T Q S Q D C T P D T S H A P A A V D S Q P N P E N E E E P R D E D W R L Q L E A E N Y

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4 206 D G H T P L H V A V I H K D A E M V R L L R D A G A D L N K P E P T

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Reception of signal (not shown) Transduction (not shown) Response – 1) ubiquitin tags IκB 2) proteases degrade IκB 1) IκB unmasks NFκB’s nuclear localization sequence 2) NFκB enters nucleus

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Figure 1. Canonical IκB’s regulation of NFκB.

Fluorescence Anisotropy Given a constant concentration of fluorescently labeled DNA (5 nM FITC DNA), as the concentration of p65/p65 increases, the tumbling speed of the molecules in solution decreases. This indicates binding between DNA and p65/p65.

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Figure 11. Fluorescence polarization analysis of varying concentrations of p65/p65

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Protein identification

X X X Y V T E D G D T A L H L A V I H Q H E P F L D F L L G F S A G T E Y L D L Q N D L G Q T A L H L A A I L G E A S T 5 10 15 20 25 30 35 40 45 50 55 60

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P D T S H A P A A V D S Q P N P E N E E E P R D E D X X X X X E A E N Y D G H T P L H V A V I H K D A E M V R L L R D A 125 130 135 140 145 150 155 160 165 170 175 180

G A D L N K P E P T C G R T P L H L A V E A Q A A S V X X L L L K A G A D P T A R M Y G G R T P L G S A L X X X X X X X 185 190 195 200 205 210 215 220 225 230 235 240

A R L L R A H G A P E P E D E D D K L S P C S S S G S D S D S D N R D E G D E Y D D 245 250 255 260 265 270 275 280

Total: 93.3% Coverage, 2.30 Redundancy

Figure 10. Peptide coverage of IκBβ collected on the Synapt G2S

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We used a Synapt mass spectrometer to perform a peptide digestion in order to identify lanes 20 and 21 from the MonoS gel as IκBβ.

We performed PCR along a gradient of melting temperatures from 71°C to 81°C to repair a mutated version of the pET11a plasmid containing the IκBβ gene. We then ran an agarose gel to determine the temperatures at which the primer annealed most successfully and adjusted future gradients accordingly.

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We then transformed the repaired plasmid into DH5α E. coli cells, which produced more of this plasmid.

Protein expression and purification Expression We transformed BL21 E. coli with the pET11a plasmid encoding the genes for IκBβ and p65. We grew these cells to OD600 = 0.6 and induced protein expression with 0.1 mM IPTG to express IκBβ and NFκB. Purification We isolated both proteins from the cells grown by: 1) Centrifuging the cells at 4,000 rpm for 20 min 2) Resuspending the pellets in lysis buffer 3) Sonicating the cellular resuspension 4) Centrifuging the result of the sonication at 12,000 rpm for 45 min 5) Running various columns with the resulting cell lysate

Columns: IκBβ = negatively charged (pI = 4.71) HIGH LOAD Q (positively charged) S200 (size exclusion) p65/p65 = positively charged SP SEPHAROSE (negatively charged) MONOS (negatively charged)

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Figure 3. Positively charged affinity column. Negatively charged columns function inversely, while Size Exclusion columns filter molecules based on size.

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Figure 4. SDS-PAGE of IκBβ HLQ fractions.

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Figure 5. SDS-PAGE of IκBβ S200 fractions.

Figure 6. Chromatogram of IκBβ S200 fractions.

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Figure 7. SDS-PAGE of p65/p65 SP Sepharose fractions.

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Figure 9. Chromatogram of p65/p65 MonoS fractions.

Figure 8. SDS-PAGE of p65/p65 MonoS fractions.

Sequence and Structure of IκBβ