PMC

P67 stimulates Cdk5/p35 activity but not Cdk5/p25 activity in an in vitro assay. The assay consists of active Cdk5/p35 or active Cdk5/p25 (5 ng) and histone H1 as a substrate (20 μg), bacterially expressed GST-p67 (1.5 μg), and [γ³²P]ATP (0.05 mM). The results represent the values of three experiments and are expressed as ±SEM. ***p < 0.001.

Diagram illustrating the hypothesis to explain the P5 specific inhibition of Cdk5/p25 in vivo. The recovery neuronal state after P5/TFP5 treatment. In recovery, TFP5 entering the neuron preferentially interacts with the endogenous Cdk5/p35 multimeric complex, which prevents peptide association with catalytic sites on Cdk5, whereas in neurons with hyperactive Cdk5/p25, TFP5 readily competes with p25 on Cdk5 catalytic sites and inhibits kinase activity.

The TFP5 peptide inhibits Cdk5/p35 and Cdk5/p25 in vitro kinase assays. In an in vitro pad assay with histone as substrate, the TFP5 peptide inhibited Cdk5/p35 and Cdk5/p25 activities at 74 and 75%, respectively, at 5 μM concentration. Note that the scrambled peptide (Scb) at 10 μM has no effect on kinase activities. The results represent the value of three experiments and are expressed as ±SEM. **p < 0.01.

Cdk5/p25 activity is specifically inhibited by TP5 peptide in transfected HEK 293 cells. HEK 293 cells were transfected with Cdk5/p35 or Cdk5/p25. The cells were then incubated in different concentrations of TP5 (0–800 nm) for 24 h, washed, and subjected to a Cdk5 immunoprecipitation procedure. (A) The respective Cdk5 immunoprecipitates were assayed for kinase activity with histone as substrate. Autoradiographs of the respective assays. H1, total histone; pH1, phospho-histone. (B). The identical lysates were used for Cdk5 kinase pad assays. Quantified results of three experiments (± SEM). *p < 0.05.

P67 specifically protects Cdk5/p35 from inhibition by the TFP5 peptide in vitro. The assay consists of active Cdk5/p35 or active Cdk5/p25 (5 ng), histone H1 as substrate (20 μg), bacterially expressed GST-p67 (0.375 μg), TFP5 at three concentrations (0.5, 1.0. and 2.0 μM), and [γ³²P]ATP (0.05 mM). (A, C) Top, autoradiographs; bottom, Coomassie-stained gels of the kinase assays. (B, D) Mean densities of histone phosphorylation in the autoradiographs measured using ImageJ software. Results represent values from four experiments and are expressed as ±SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Identification of GST-p10 proteins pulled down from the rat brain lysate. Proteins were isolated from rat brain extracts in pull-down experiments using GST or GST-p10 and separated by SDS–PAGE on 4–20% gels and immunodetected with Western blot (WB) analysis using respective antibodies as shown (n = 3). (A) GST control. Except for the expression of tau, β-actin, and β-tubulin, very few proteins were pulled down from rat brain lysate with GST alone. (B) In contrast, the GST-p10 pull down resulted in the expression of cytoskeletal proteins NFL, tubulin, actin, and tau and included expression of p67, p35, GFAP, and syntaxin.

P67 preferentially binds to CDK5/p35 in the presence of TFP5 in cotransfected HEK 293 cells. HEK 293 cells were cotransfected with Cdk5/p35 (myc-his)/p67 or Cdk5/p25 (myc-his)/p67 and incubated in the presence of different concentrations of TFP5 peptide for 24 h. Lysates were then prepared and subjected to Western blotting to visualize protein expression of transfected plasmids with respective antibodies. (A) Expression of different proteins in the lysates using p67, p35/p25, Cdk5, and β-actin antibodies. (B) Immunoprecipitation using 500 μg of proteins (p35 myc-his and p25 myc-his) with monoclonal his-tag antibody detected with p67, p35, Cdk5, and β-actin antibodies, respectively, after Western blotting. (C) Quantification of immunoblots of p67 and p35 antibodies, respectively, from three experiments (± SEM). **p < 0.01.

P67 binds to Cdk5/p35, cytoskeletal proteins and synaptic proteins in a rat brain lysate. (A) Rat brain lysate was immuno­precipitated (IP) with p67 antibody and the bound proteins, eluted from washed beads, were immunodetected with Western blot (WB) analyses using respective antibodies as shown. See Materials and Methods for antibody concentrations (n = 3). (B) A reciprocal p35 IP of a similar rat brain lysate shows bound proteins similar to those in a p67 coimmunoprecipitation (n = 3). Rabbit IgG was used as controls for each antibody shown, and, except for tubulin, no proteins were immunoprecipitated. An example of control blots is shown in lane 1. For lane 10 (Ctrl) in A and lane 8 (Ctrl) in B, tubulin was detected with anti-tubulin antibody.

TFP5 competes specifically with p25 binding to Cdk5 without affecting p35-Cdk5 binding in transfected HEK 293 cells. HEK 293 cells were transfected with Cdk5/p35 or Cdk5/p25 and incubated in 800 nM TFP5 or scrambled peptide control (SCB) for 24 h. (A, B) Lysates were prepared for Western blots to assess levels of expression (A) and subjected to a Cdk5 immunoprecipitation with J-3 antibody to determine the extent of p25 and p35 binding to Cdk5 by Western blots using the respective antibodies (B). (C) Mean density scans of lanes 3–8 were carried out with the National Institutes of Health ImageJ (n = 3, ±SEM, *p < 0.05)

TFP5 inhibited p35/Cdk5 activity in cortical neurons transfected with p67 siRNA. P67 siRNA and control siRNA were transfected into rat primary cultured cortical neurons (on day 7). Cells were then incubated with TFP5 (200 nM) or SCB (200 nM) peptides for 72 h. Cells treated with roscovitine (25 μM) for 2 h were used as a positive control. Cells were harvested and subjected to Western blotting and Cdk5 kinase assay. (A) Western blots showing the expression of p67 and p35 in the cells transfected with p67 siRNA and treated with TFP5 or SCB peptides. P67 expression was reduced (lanes 4–7) in the presence of p67 siRNA. P67 siRNA did not affect p35 or Cdk5 expression (lanes 1–7). (B) Radiographs of Cdk5 IP activity in cells transfected with p67 siRNA were inhibited by TFP5 and unaffected by scrambled peptide (compare lanes 4 and 5). Roscovitine showed robust inhibition of Cdk5 activity (lane 7). (C) Activities of the same lysates determined in pad assays. Data are expressed as mean of three separate experiments. *p < 0.01.

The p10 N-terminal domain of p35 preferentially binds multimeric proteins in cortical neurons transfected with p35 myc-his. Rat cortical neurons at 5 days in vitro were transfected with p35 myc-his or p25 myc-his plasmid. After 48 h, lysates were prepared and subjected to SDS–PAGE using 30 μg of lysate for each lane. (A) Protein expression was analyzed using three different antibodies to confirm p35 and p25 expression with p35 (C-19), myc, and his antibodies, respectively. Arrowhead and arrow represent the expression of myc-his p35 and myc-his p25, respectively. (B–F). Both p35 and p25 lysates were used at 500-μg protein concentration for immunoprecipitation with mouse monoclonal myc antibody. Input represents expression level of each protein. After SDS–PAGE, Western blotting was performed using different polyclonal antibodies as follows: p67 (B), PSD95 (C), tau (D), GFAP (E), and syntaxin 1 (F). Data were analyzed with Prism 5.0 software. Tukey’s multiple comparisons test was used. Differences with *p < 0.05 were considered significant. The quantification of bands was done using ImageJ software. The results represent the value of three experiments and are expressed as ±SEM. **p < 0.01 and ***p < 0.001.

P5-biotin pull-down assays of rat brain lysates and transfected HEK293 cells show P5 binding to Cdk5, p35, and p67. (A) Two different concentrations of P5-biotin were used in affinity binding assays with Scb (scrambled peptide) as negative control. Protein extracts (500 μg in 500 μl) from mouse brain tissues were incubated with different concentrations (0.5 and 0.25 mg/ml) of P5-biotin, scrambled-biotin, or P5 without biotin overnight at 4°C. On the second day, streptavidin magbeads (50 μl) were added to the lysates for 2 h at 4°C. After gentle spinning, streptavidin magbeads were collected and washed three times. The proteins were eluted from streptavidin magbeads by using sample buffer and heating at 96°C for 5 min. The eluted proteins were used for Western blot analysis, where each lane is immunoassayed with the three antibodies. (B) P5-biotin binds directly to both Cdks and p67. Expression of HEK293 cell lysates (25 μg) as transfected with empty vector (EV; lane 1), Cdk5 (lane 2), or p67 (lane 3). A 500-μg amount of each lysate was mixed with 0.5 mg of P5-biotin, followed by end-to-end rotation at 4°C overnight. On the next morning, the complex was pulled down with streptavidin beads. After washing three times with l× Tris-buffered saline, the beads were subjected to SDS–PAGE and Western blot analysis using anti-Cdk5 and anti-p67 antibodies in each lane. The EV negative control indicates that endogenous p67 and Cdk5 in HEK 293 cells are either absent or at levels too low to be detected.