Kenneth B. Rank

Tau Phosphorylation at Serine 396 and Serine 404 by Human Recombinant Tau Protein Kinase II Inhibits Tau's Ability to Promote Microtubule Assembly

In Alzheimers disease, hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies and fails to promote microtubule assembly. Dysregulation of the brain-specific tau protein kinase II is reported to play an important role in the pathogenesis of Alzheimers disease (Patrick, G. N., Zukerberg, L., Nikolic, M., De La Monte, S., Dikkes, P., and Tsai, L.-H. (1999) Nature 402, 615–622). We report here that in vitro phosphorylation of human tau by human recombinant tau protein kinase II severely inhibits the ability of tau to promote microtubule assembly as monitored by tubulin polymerization. The ultrastructure of tau-mediated polymerized tubulin was visualized by electron microscopy and compared with phosphorylated tau. Consistent with the observed slower kinetics of tubulin polymerization, phosphorylated tau is compromised in its ability to generate microtubules. Moreover, we show that phosphorylation of microtubule-associated tau results in taus dissociation from the microtubules and tubulin depolymerization. Mutational studies with human tau indicate that phosphorylation by tau protein kinase II at serine 396 and serine 404 is primarily responsible for the functional loss of tau-mediated tubulin polymerization. These in vitroresults suggest a possible role for tau protein kinase II-mediated tau phosphorylation in initiating the destabilization of microtubules.

Direct interaction of soluble human recombinant tau protein with Aβ 1–42 results in tau aggregation and hyperphosphorylation by tau protein kinase II

We report here that aggregated β‐amyloid (Aβ) 1–42 promotes tau aggregation in vitro in a dose‐dependent manner. When Aβ‐mediated aggregated tau was used as a substrate for tau protein kinase II (TPK II), an 8‐fold increase in the rate of TPK II‐mediated tau phosphorylation was observed. The extent of TPK II‐dependent tau phosphorylation increased as a function of time and Aβ 1–42 concentration, and hyperphosphorylated tau was found to be decorated with an Alzheimers disease‐related phosphoepitope (P‐Thr‐231). In HEK 293 cells co‐expressing CT‐100 amyloid precursor protein and tau, the release of Aβ 1–42 from these cells was impaired. Taken together, these in vitro results suggest that Aβ 1–42 promotes both tau aggregation and hyperphosphorylation.

A scintillation proximity assay for studying inhibitors of human tau protein kinase II/cdk5 using a 96-well format.

Dysregulation of the brain-specific tau protein kinase II (TPK II)/cdk5 is reported to play an important role in the pathogenesis of Alzheimers disease. We report here a quantitative scintillation proximity assay (SPA), which is suitable for determining TPK II/cdk5 activity and its inhibition. It depends upon the phosphorylation of a synthetic histone-based peptide substrate (PKTPKKAKKL), which has been biotinylated at its C-terminus. When this biotinylated peptide is incubated with [gamma-33P] ATP and TPK II/cdk5 under defined assay conditions, product formation is linear with respect to time and enzyme concentration. The production of [33P] phosphorylated peptide is inhibited in the presence of a known TPK II/cdk5 inhibitor but is unaffected in the presence of 1% DMSO. A signal-to-noise ratio of 16:1 was obtained in a 60-min assay with an intra-assay variability of <10% in the 96-well microtiter format. The TPK II/cdk5 SPA is very robust, sensitive and simple to perform.

Mechanism of resistance to U-90152S and sensitization to L-697,661 by a proline to leucine change at residue 236 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase

Bisheteroarylpiperazines (BHAPs) are highly specific inhibitors of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT). BHAP‐resistant HIV‐1 is sensitized to other classes of nonnucleoside RT inhibitors and this has been primarily attributed to a proline‐to‐leucine substitution at amino acid 236 (P236L) of HIV‐1 RT. To understand the basis for the in vitro sensitization‐resistance phenomenon, single base pair mutations at amino acid P236 in HIV‐1 RT were introduced to obtain P236L, P236T, P236H, P236R, and P236A HIV‐1 RT mutants. Active HIV‐1 RT mutants H235W, D237T, and H235W/D237T/T240K, containing substitutions from HIV‐2 RT, were also cloned, expressed, and purified. Three BHAPs (U‐88204E, U‐87201E, and U‐90125S) and the pyridinone L‐697,661 were selected to quantitatively assess the effects of these amino acid substitutions on sensitization to L‐697,661 and resistance to the BHAPs. The HIV‐1 RT mutants bearing single (H235W; D237T) or multiple (H235W/D237T/T240K) HIV‐2 RT substitutions around the conserved P236 conferred little resistance or sensitization to these RT inhibitors. The inhibition profiles of the P236 HIV‐1 RT mutants demonstrated a direct correlation between sensitization to L‐697,661 and resistance to the BHAPs. These results suggest alterations in the shape of the binding pocket as the mechanism by which the P236L mutation confers resistance to the BHAPs and sensitization to L‐697,661.

Simultaneous mutations at Tyr-181 and Tyr-188 in HIV-1 reverse transcriptase prevents inhibition of RNA-dependent DNA polymerase activity by the bisheteroarylpiperazine (BHAP) U-90152s

The replacement of either Tyr‐181 or Tyr‐188 of human immunodeficiency virus type 1 (HIV‐1) reverse transcriptase (RT) by the corresponding HIV‐2 RT amino acids Ile‐181 or Leu‐188 is known to result in active mutant enzymes (Y181I; Y188L) with virtual loss of sensitivity towards three structural classes of nonnucleoside RT inhibitors; L‐697,661, nevirapine, and TIBO R82913. The bisheteroarylpiperazine (BHAP) U‐90152S, a highly specific inhibitor (IC50, 0.29 ± 0.01 μM) of HIV‐1 RT, inhibited the recombinant Y181I and Y188L HIV‐1 RT mutants with IC50 values of 3.6 ± 0.15 μM and 0.71 ± 0.02 μM, respectively. Construction and in vitro analysis of double mutants Y181I/Y188L and Y181C/Y188L of HIV‐1 RT showed > 150‐fold resistance to U‐90152S. An HIV‐2 mutant containing amino acids 176–190 from HIV‐1 RT acquired full sensitivity to U‐90152S (IC50, 0.29 ± 0.01 μM). It is concluded tha simultaneous mutations at Tyr‐181 and Tyr‐188 of HIV‐1 RT promotes resistance to U‐90152S.