Fig. 2.

Dimerization of the kinase domain is autoinhibitory. (A) Radiometric autophosphorylation assay of PKD1^KD and PKD3^KD. Error bars are the SD of two to four biologically independent experiments. Inset: Data points represent a reaction plateau at corresponding PKD^KD concentrations. Bottom: Representative autoradiograph of PKD1^KD samples compared to that of loading control (silver stain). (B) Substrate phosphorylation kinetics of PKD1^KD compared to those of ULD-mediated PKD1^KD dimers with either (Gly)₅ or (Gly)₁₀ linkers between the ULD and kinase domains. Assay performed at concentrations corresponding to monomeric PKD1^KD. Error bars are the SD of n biologically independent experiments. Table indicates the number (n) of replicates and the values for k_cat derived from a linear regression in the linear range. (C) Autophosphorylation kinetics of wild-type PKD1^KD compared to those of monomeric PKD1^KD E787K at low (50 nM) and high (5 μM) concentrations. Error bars are the SD of three biologically independent experiments. (D) PKD1 activation loop phosphorylation in HEK293T cells. Wild-type PKD1 (black) and PKD1 E787K (cyan).