Identification of the kinase responsible for the hyperphosphorylation of paratarg-7. (A) Inhibition of kinases. LCLs derived from a healthy donor carrying wild-type paratarg-7 (row A) or a patient carrying hyperphosphorylated paratarg-7 (row B) were cultured in the presence of inhibitors (dissolved in DMSO) at the indicated concentrations. Lysates were analyzed by IEF and stained with anti-paratarg-7. Lane 1 indicates LCLs from healthy donor and patient, respectively, cultured with addition of DMSO (1:10⁶) only. Hyperphosphorylation was inhibited by incubation with staurosporine (2.5μM, lane 2), ellagic acid (10μM, lane 10), and wortmannin (5nM, lane 11). No effect was observed by incubation with, lanes 3 to 9: H89 (50nM), Akt1,2 inhibitor (200nM), purvalanol (100nM), SL327 (200nM), SP600125 (100nM), SB202190 (50nM), and ZM447439 (1μM). As controls, lysates of healthy donor (lane 12), patient (lane 13), and recombinant paratarg-7 in Escherichia coli (lane 14) were also analyzed. Panel A is composed of 4 separate IEF gels. (B) Inhibition of paratarg-7 hyperphosphorylation by PKCζ pseudosubstrate in LCL derived from patients with pP-7. IEF followed by immunodetection of paratarg-7 in LCLs derived from healthy donor (lane 1) and from a patient (lane 2) as control. LCLs derived from a patient with pP-7 were cultured without inhibitors (lane 3) or in the presence of bisindolylmaleimide I (6μM, lane 4) or PKCζ pseudosubstrate (50μM, lane 5). (C) Demonstration of the direct interaction of Ser17 of paratarg-7 with PKCζ: only mutated fragments containing Ser17 interact with PKCζ. No interaction was detected when Ser was replaced by Ala. wt indicates unmutated paratarg-7 fragment spanning aa 1 to 60; Ser17Ala, fragment spanning aa 1 to 60 with Ser17 replaced by Ala; Ser21Ala, fragment spanning aa 1 to 60 with Ser21 replaced by Ala; Ser17Ala Ser21Ala, fragment spanning aa 1 to 60 with both serines replaced by Ala; –, control (carrier of wild-type paratarg-7 wtP-7); +, carrier of hyperphosphorylated paratarg-7 pP-7. (D) Coimmunoprecipitation of paratarg-7 and PKCζ in cell lines (HEK293, LP1) and LCLs derived from patients (P18, P39) and healthy donor (HD7). In the left portion of the panel, precipitation of the complex was done using anti-STOML2 (BD Biosciences) followed by detection of PKCζ in the redissolved precipitate using anti-PKCζ. In the right part of the panel, precipitation was done with anti-PKCζ followed by detection with anti-STOML2. – indicates precipitation was performed with an irrelevant secondary Ab; and +, as described before. (E) In vitro phosphorylation of paratarg-7 with PKCζ. In an in vitro experiment purified recombinant wild-type paratarg-7 was incubated with recombinant PKCζ in the presence or absence of PKCζ pseudosubstrate. The figure shows an IEF followed by immunodetection of paratarg-7. 1 indicates recombinant E coli paratarg-7; 2, recombinant E coli paratarg-7 (0.5 μg) phosphorylated with recombinant PKCζ (0.5 μg) in the absence or 3, presence of PKCζ pseudosubstrate (50μM).