Renaturation of Phosphorylase Kinase Activity From Sodium Dodecyl Sulfate-Polyacrylamide Gels

Phosphorylase kinase activity is renatured and detected in situ following electrophoresis of the denatured holoenzyme in a sodium dodecyl sulfate-polyacrylamide gel containing phosphorylase b that has been included in the gel polymerization according to the method of R. L. Geahlen et al [(1986) Anal. Biochem. 153, 151-158]. Among the enzyme\u27s four subunits, only γ is catalytically active. When extract of rabbit muscle is electrophoresed and renatured in a similar manner, the phosphorylase-conversion activity is also associated only with a protein band that comigrates with the γ subunit of phosphorylase kinase. This suggests that the γ subunit of phosphorylase kinase may be the sole activity in rabbit muscle responsible for the phosphorylation of phosphorylase b. In an alternative method for the renaturation of activity from conventional sodium dodecyl sulfate-polyacrylamide gels, the subunits of the enzyme are visualized using 2.5 m KCl, excised from the gel, and eluted by diffusion into buffer containing sodium dodecyl sulfate, which is subsequently removed by acetone precipitation of the eluted subunits. Catalytic activity is recovered when the acetone precipitate of the extracted γ subunit is dissolved in 6 m guanidine hydrochloride and diluted 50-fold into an activity assay. Inclusion of eluted α and β subunits in the assay inhibits the activity of the γ subunit, which supports our previous finding that the α and/or β subunits suppress the activity of the catalytic γ subunit [H. K. Paudel and G. M. Carlson (1987) J. Biol. Chem. 262, 11912-11915]

14-3-3ζ Mediates Tau Aggregation in Human Neuroblastoma M17 Cells

<div><p>Microtubule-associated protein tau is the major component of paired helical filaments (PHFs) associated with the neuropathology of Alzheimer’s disease (AD). Tau in the normal brain binds and stabilizes microtubules. Tau isolated from PHFs is hyperphosphorylated, which prevents it from binding to microtubules. Tau phosphorylation has been suggested to be involved in the development of NFT pathology in the AD brain. Recently, we showed that 14-3-3ζ is bound to tau in the PHFs and when incubated <i>in vitro</i> with 14-3-3ζ, tau formed amorphous aggregates, single-stranded straight filaments, double stranded ribbon-like filaments and PHF-like filaments that displayed close resemblance with corresponding ultrastructures of AD brain. Surprisingly however, phosphorylated and non-phosphorylated tau aggregated in a similar manner, indicating that tau phosphorylation does not affect <i>in vitro</i> tau aggregation (Qureshi <i>et al</i> (2013) Biochemistry 52, 6445–6455). In this study, we have examined the role of tau phosphorylation in tau aggregation in cellular level. We have found that in human M17 neuroblastoma cells, tau phosphorylation by GSK3β or PKA does not cause tau aggregation, but promotes 14-3-3ζ-induced tau aggregation by destabilizing microtubules. Microtubule disrupting drugs also promoted 14-3-3ζ-induced tau aggregation without changing tau phosphorylation in M17 cell. <i>In vitro</i>, when incubated with 14-3-3ζ and microtubules, nonphosphorylated tau bound to microtubules and did not aggregate. Phosphorylated tau on the other hand did not bind to microtubules and aggregated. Our data indicate that microtubule-bound tau is resistant to 14-3-3ζ-induced tau aggregation and suggest that tau phosphorylation promotes tau aggregation in the brain by detaching tau from microtubules and thus making it accessible to 14-3-3ζ.</p></div

Cyclin-dependent protein kinase 5 primes microtubule-associated protein tau site-specifically for glycogen synthase kinase 3β

In the preceding paper, we showed that GSK3β phosphorylates tau at S202, T231, S396, and S400 in vivo. Phosphorylation of S202 occurs without priming. Phosphorylation of T231, on the other hand, requires priming phosphorylation of S235. Similarly, priming phosphorylation of S404 is essential for the sequential phosphorylation of S400 and S396 by GSK3β. The priming kinase that phosphorylates tau at S235 and S404 in the brain is not known. In this study, we find that in HEK-293 cells cotransfected with tau, GSK3β, and Cdk5, Cdk5 phosphorylates tau at S202, S235, and S404. S235 phosphorylation enhances GSK3β-catalyzed T231 phosphorylation. Similarly, Cdk5 by phosphorylating S404 stimulates phosphorylation of S400 and S396 by GSK3β. These data indicate that Cdk5 primes tau for GSK3β in intact cells. To evaluate if Cdk5 primes tau for GSK3β in mammalian brain, we examined localizations of Cdk5, tau, and GSK3β in rat brain. We also analyzed the interaction of Cdk5 with tau and GSK3β in brain microtubules. We found that Cdk5, GSK3β, and tau are virtually colocalized in rat brain cortex. When bovine brain microtubules are analyzed by FPLC gel filtration, Cdk5, GSK3β, and tau coelute within an ∼450 kDa complex. From the fractions containing the ∼450 kDa complex, tau, Cdk5, and GSK3β co-immunoprecipitate with each other. In HEK-293 cells transfected with tau, Cdk5, and GSK3β in different combinations, tau binds to Cdk5 in a manner independent of GSK3β and to GSK3β in a manner independent of Cdk5. However, Cdk5 and GSK3β bind to each other only in the presence of tau, suggesting that tau connects Cdk5 and GSK3β. Our results suggest that in the brain, tau, Cdk5, and GSK3β are components of an ∼450 kDa complex. Within the complex, Cdk5 phosphorylates tau at S235 and primes it for phosphorylation of T231 by GSK3β. Similarly, Cdk5 by phosphorylating tau at S404 primes tau for a sequential phosphorylation of S400 and S396 by GSK3β

Co-expression of 14-3-3ζ and tau in human neuroblastoma cells causes formation of cytoplasmic thioflavin S positive inclusions.

<p>Cells transfected with either Flag-tau or Flag-tau and Myc-14-3-3ζ were fixed and then stained with thioflavin S (green) followed by either anti-Flag for tau (red) or anti-Myc for 14-3-3ζ (red). <i>A</i>, cells transfected with tau alone stained for tau (red), Thioflavin S (green) and DAPI (nucleus). <i>B</i> and <i>C</i>, cells co-transfected with Flag-tau and Myc-14-3-3ζ. Scale bars 40 μM (<i>A</i>); 100 μM (<i>B</i> and <i>C</i>).</p

Immuno EM of tau aggregates formed in M17 human neuroblastoma cells expressing Flag-tau, Myc-14-3-3ζ, HA-GSK3β and Myc-PKA in various combination.

<p>Pellets obtained by centrifugation assay from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160635#pone.0160635.g004" target="_blank">Fig 4<i>B</i></a> were analyzed by Immuno-EM as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160635#pone.0160635.g003" target="_blank">Fig 3<i>E</i></a>. Scale bar 100 nm.</p

Tau and 14-3-3ζ forms a high molecular weight complex when co-expressed in M17 human neuroblastoma cells.

<p>Cells transfected with either Flag-tau or Myc-14-3-3ζ or co-transfected with Flag-tau and Myc-14-3-3ζ were subjected to OmniPrep density gradient centrifugation. Fractions were collected and analyzed by Western blotting and immunoprecipitation. <i>A</i>, Western blot of fractions corresponding to cells transfected with Flag-tau alone. <i>B</i>, Western blot of fractions corresponding to cells transfected with Myc-14-3-3ζ alone. <i>C</i>, Western blot of samples corresponding to cells co-transfected with Flag-tau and Myc-14-3-3ζ. Based on blot band intensities, the relative amount of Peak 1 (sum of fractions 1–4) and peak 2 (sum of fractions 5–8) was calculated and is expressed as the % of total (sum of fractions 1–8). <i>D</i>, Immunoprecipitation. Immunoprecipitation was carried out using fraction # 6 from panel <i>C</i>.</p

Microtubules protect tau from 14-3-3ζ-induced aggregation <i>in vitro</i>–Microtubules sedimentation assay was performed as described in Materials and Methods.

<p><i>A</i>, Western blots of microtubule sedimentation assay performed in the presence of GTP/Mg²⁺/taxol followed by the addition of GST. <i>B</i>, Western blots of microtubules sedimentation assay in the presence of GTP/ Mg²⁺/taxol followed by the addition of 14-3-3ζ. <i>C</i>, Immuno EM of P2 from microtubule sedimentation assay in the presence of GTP/ Mg²⁺/taxol followed by the addition of 14-3-3ζ. <i>D</i>, Western blots of microtubule sedimentation assay in the absence of GTP/ Mg²⁺/taxol followed by the addition of GST. <i>E</i>, Western blots of microtubule sedimentation assay in the absence of GTP/ Mg²⁺/taxol followed by the addition of 14-3-3ζ. <i>F</i>, Immuno EM of P2 from microtubule sedimentation assay in the absence of GTP/ Mg²⁺/taxol followed by the addition of 14-3-3ζ. Scale bars 100 nm.</p

Inverse correlation between microtubule stability and 14-3-3ζ-induced tau aggregation in M17 human neuroblastoma cells.

<p>Samples from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160635#pone.0160635.g004" target="_blank">Fig 4<i>A</i></a> were analyzed by Western blot analysis for microtubules. The relative amount of tubulin was calculated from normalizing the tubulin band of each sample with the corresponding actin band of that sample. Likewise, the relative amounts of Ac-Tub (Ac-tubulin) and Tyr-Tub (Tyr-tubulin) were calculated by normalizing Ac-Tub band or Tyr-Tub band with corresponding β-tubulin band. The relative amount of aggregated tau is from panel <i>B</i> of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160635#pone.0160635.g004" target="_blank">Fig 4</a>. Values in the bar graph are the average ± S.E. of three independent experiments. *<i>p</i>< 0.05 against the cells transfected with Flag-tau alone. <i>F</i>, Correlation. Data for microtubule stability and aggregated tau are from panel <i>C</i> and <i>E</i>, respectively. Plot was generated by using values from lanes 1, 2, 4 and 6 only.</p

Phosphorylation promotes 14-3-3ζ-induced tau aggregation in M17 human neuroblastoma cells–M17 human neuroblastoma cells co-transfected with Flag-tau, Myc-14-3-3ζ, HA-GSK3β and Myc-PKA in various combinations were analyzed by Western blotting for tau phosphorylation and then by centrifugation assay for tau aggregation.

<p><i>A</i>, Western blot analysis. PHF-1 and pS214 blots represent tau phosphorylated at Ser^396/404 and Ser²¹⁴, respectively. The ser^396/404 site is phosphorylated by GSK3β whereas the ser²¹⁴ site is phosphorylated by PKA. Based on tau band intensities the relative amount of phosphorylated tau was determined. The relative amount of phosphorylated tau was determined by normalizing the phosphorylated tau band intensity by respective band intensity of total tau. Values with ±SE are the average of three determinations. *<i>P</i>< 0.05 with respect to control cells expressing tau alone. <i>B</i>, Centrifugation assay. The centrifugation assay was performed as described in the Materials and Methods. S and P indicate supernatant and pellet, respectively. Based on Flag-tau blot band intensity, the relative distribution of tau and the relative amount of aggregated tau in the indicated fractions were determined as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160635#pone.0160635.g002" target="_blank">Fig 2<i>A</i></a>. The relative amount of aggregated tau is expressed as fold of cells expressing tau and 14-3-3ζ. Values in the bar graph are mean ± S.E. and are from three independent experiments. *<i>P</i> < 0.05 with respect to cells expressing tau and 14-3-3ζ.</p