FIGURE 4.

Amino acid substitution mutants of p35 AD and p39 AD. A, amino acid sequences of α5-α6 of p35 and p39. The three amino acids that are involved in hydrogen bond formation are highlighted. Substitution mutants used are shown below. B, p35 AD (WT) or one of the single amino acid mutants (D259Q, N266Q, and S270P) were co-expressed with Cdk5 in HEK293 cells. Their complexes with Cdk5 were immunoprecipitated with anti-FLAG and detected by immunoblotting with anti-FLAG (p35 AD or mutants) and anti-Cdk5 (upper panel). The kinase activity was measured using histone H1 (H1) and [γ-³²P]ATP as substrates in the absence (−) or presence (+) of 1% Nonidet P-40 (lower panel). C, p35 AD (WT) or the double amino acid mutants (D259Q/N266Q, D259Q/S270P, and N266Q/S270P) were co-expressed with Cdk5 in HEK293 cells. Their complexes with Cdk5 were immunoprecipitated and detected by immunoblotting with anti-FLAG (p35 AD or mutants) and anti-Cdk5 (upper panel). The kinase activity was measured using histone H1 (H1) and [γ-³²P]ATP as substrates in the absence (−) or presence (+) of 1% Nonidet P-40 (lower panel). D, p35 AD or its triple amino acid mutant (p35 AD-TM) was co-expressed with Cdk5 in HEK293 cells. Immunoblotting shows the expression of p35 AD (WT) or p35 AD-TM (TM) and Cdk5. After washing the immunoprecipitates in the absence (−) or presence (+) of 1% Nonidet P-40, p35 AD and Cdk5 (Cdk5) were examined by immunoblotting, and the kinase activity (H1) was measured using histone H1 and [γ-³²P]ATP as substrates. E, expression of p35 (WT) or p35-TM (TM) and Cdk5 in HEK293 cells (upper panel). After washing the immunoprecipitates in the absence (−) or presence (+) of 1% Nonidet P-40, p35 or Cdk5 was examined by immunoblotting, and the kinase activity (H1) was measured using histone H1 and [γ-³²P]ATP as substrates. Quantification is shown as the relative ratio to the kinase activity of Cdk5-p35 in the absence of Nonidet P-40 (means ± S.E., n = 3).