Report

A Signaling Complex of Ca2+-Calmodulin-Dependent Protein Kinase IV and Protein Phosphatase 2A

See allHide authors and affiliations

Science  22 May 1998:
Vol. 280, Issue 5367, pp. 1258-1261
DOI: 10.1126/science.280.5367.1258

Figures

  • Figure 1

    Identification of a CaMKIV-PP2A complex. (A) Coimmunoprecipitation of PP2A with CaMKIV. Immunoprecipitations from Jurkat T cell extracts were performed as described (7) with a rabbit polyclonal antibody raised against the COOH-terminal 17 residues of human CaM KIV (Imm.) or preimmune serum (Preimm.). Immune complexes were subjected to immunoblot analysis with a monoclonal antibody to the C subunit of PP2A (15). (B) Isolation of PP2A with a GST-CaMKIV fusion protein (9). Rat brain soluble extracts were incubated with buffer or wild-type GST-CaMKIV fusion protein (10 μg) for 3 hours at 4°C and then purified with glutathione-Sepharose (17). The resin was extensively washed and bound proteins were eluted with 20 mM glutathione, resolved by SDS-PAGE, and subjected to immunoblot analysis with antibodies to the C and Bα regulatory subunits of PP2A. (C) Binding to calmodulin-Sepharose. Fractions containing proteins of large molecular mass (250 to 700 kD) from gel filtration of rat brain soluble extracts were incubated with calmodulin-Sepharose in the presence (+Ca2+) or absence (–Ca2+) of 5 mM CaCl2 (36). Bound proteins were eluted with 15 mM EGTA, resolved by SDS-PAGE, and subjected to protein immunoblotting with antibodies to the indicated PP2A subunits. (D) Elution of CaMKIV and PP2A from a Superdex-200 gel filtration column after sequential fractionation of brain extracts on phenyl-Sepharose, calmodulin-Sepharose, and Mono Q columns (14). Presented is an immunoblot of fractions from the final gel filtration column showing CaMKIV and the C and Bα subunits of PP2A. Although not shown, the A subunit of PP2A was also present. (E) Calmodulin overlay was performed on fraction 23 of the gel filtration column in the presence of 1 mM Ca2+ or 1 mM EGTA (15).

  • Figure 2

    Independence of association of CaMKIV and PP2A from enzyme activity. (A) Isolation of PP2A with wild-type and mutant GST-CaMKIV fusion proteins (9,17). Rat brain soluble extracts were incubated with the indicated GST fusion proteins (10 μg) and analyzed as described in Fig. 1. Data shown are from a single experiment repeated two to six times with various wild-type and mutant GST-CaMKIV fusion proteins. (B) Isolation of CaMKIV with microcystin-Sepharose. Rat brain or Jurkat T cell soluble extracts were incubated with microcystin-Sepharose in the absence (–) or presence (+) of the PP2A inhibitor microcystin (19). Bound proteins were eluted with SDS-PAGE sample buffer and subjected to immunoblot analysis with antibodies to the C subunit of PP2A and to CaMKIV. The rat brain immunoblot (middle) was stained with Ponceau S (left) to compare proteins eluted from microcystin-Sepharose (15). The locations of the A, Bα, and C subunits of PP2A are marked by arrows. Results are representative of three independent experiments.

  • Figure 3

    Dephosphorylation of CaMKIV by PP2A. A CaMKIV-PP2A preparation (Fig. 1, gel filtration fraction 23) was incubated for 15 min at 30°C in the presence of 10 mM MgCl2, 100 μM ATP, and [γ-32P]ATP (2000 dpm/pmol) and the indicated components (+); CaMKK (0.53 ng/μl) 3 mM CaCl2, 1 μm calmodulin, and 1 μm okadaic acid. EGTA (25 mM) was added, and the incubation was continued an additional 30 min. Reactions were stopped with sample buffer and subjected to SDS-PAGE and autoradiography.

  • Figure 4

    Regulation of CREB activity in Jurkat T cells. (A) Enhancement of CaMKIV-mediated CREB activity by small t antigen, an inhibitor of PP2A activity. Jurkat cells were transiently cotransfected with 5×(Gal4)-luciferase reporter plasmid (5 μg) and Gal4-CREB expression plasmid (5 μg) together with expression plasmids for CaMKIV (3 μg), small t (1 μg), or constitutively active protein kinase A (PKA, 0.1 μg), as indicated (37). Eighteen hours after transfection, cells were stimulated for 5 hours with buffer (control) or calcium ionophore (1 μM ionomycin) and assayed for luciferase activity. A low concentration of PKA expression plasmid cDNA (0.1 μg) was used in these experiments to achieve a small amount of luciferase activity. Increasing the amount of PKA expression plasmid resulted in a large increase in luciferase activity that also was unaffected by expression of small t (16). Bars represent the relative induction over the value for control plasmid alone, normalized for amount of protein and efficiency of transfection. The values are means ± SE (n = 4). (B) A model for Ca2+-mediated phosphorylation of CREB in T lymphocytes.

Navigate This Article