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Ca²⁺-binding proteins of cilia and infraciliary lattice of Paramecium tetraurelia: their phosphorylation by purified endogenous Ca²⁺-dependent protein kinases

¹ Department of Oncology, McArdle Lab, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA
² Department of Biochemistry, College of Agricultural and Life Science, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA

View larger version (55K): [in a new window]   Fig. 1. Purification of Ca2+-binding proteins on phenyl-Sepharose monitored by a 45Ca2+ blot overlay assay. Samples were subjected to SDS-PAGE, transferred to nitrocellulose and probed with 45Ca2+ as described in the Materials and Methods. The free Ca2+ concentration was 1.4 µM. After autoradiography (B), the membrane was stained with amido black (A). Lane 1, markers, 2 µg each; lane 2, phenyl-Sepharose load, 50 µg; lane 3, phenyl-Sepharose EGTA eluate, 20 µg; M, Mr of markers (x103).

View larger version (35K): [in a new window]   Fig. 2. Purification of PCBP-25 and CaM on MonoQ (A) and chromatographic separation of PCBP-25 and PCBP-25ß visualized by silver stain (B). Chromatography was performed as described in the Materials and Methods. In A, the fractions containing PCBP-25 determined by on-blot 45Ca2+-binding assay are indicated, as are the positions of two CaPKs and CaM. In (B), MonoQ fractions (15 µl each) were run on SDS-PAGE and silver stained as described in the Materials and Methods. The positions of Ca2+-binding proteins and the fraction numbers are indicated. M, Mr of markers (x103).

View larger version (33K): [in a new window]   Fig. 3. Immunoblot analysis of (A) antibody specificity and (B) immunocrossreactivity of PCBP-25 and PCBP-25ß. Samples were subjected to SDS-PAGE, transferred to nitrocellulose and probed with either polyclonal antibody against Chlamydomonas centrin or with monoclonal antibody 7A9 as indicated. (A) Lane C, Chlamydomonas whole cell, 15 µg; lane P, quick-killed Paramecium, 20 µg. (B) MonoQ fractions (20 µl) containing PCBP-25. Fraction numbers are indicated at the bottom of the blot. M, Mr of markers (x103).

View larger version (53K): [in a new window]   Fig. 4. Immunodetection of PCBP-25 in subciliary fractions. Subciliary fractions were generated as described in the Materials and Methods. (A) Coomassie-stained SDS-PAGE, (B) immunoblots with antibody against centrin and (C) with monoclonal antibody 7A9. The same amount of protein (15 µg) was loaded in each lane. Lane 1, whole cilia; lane 2, Triton-extracted ciliary membrane; lane 3, axoneme; lane 4, dynein fraction released by high salt from axoneme; lane 5, axoneme after high-salt extraction. M, Mr of markers (x103).

View larger version (81K): [in a new window]   Fig. 5. Immunodetection of PCBP-25 in purified dynein fractions. Paramecium 22S and 12S dyneins were purified by sucrose gradient sedimentation from the high-salt extract (0.6 M KCl) of demembranated axonemes. Ponceau S-stained SDS-PAGE (A), immunoblots with antiserum against centrin or with monoclonal antibody 7A9 (B). The same amount of protein (10 µg) was loaded in each lane. Lane 1, 22S dynein; lane 2, 12S dynein; M, Mr of markers (x103).

View larger version (43K): [in a new window]   Fig. 6. Immunodetection and 45Ca2+ binding of PCBP-25 in EGTA-extracted supernatant from infraciliary lattice (ICL) preparation. Purification and EGTA extraction (10 mM) of ICL were performed as described in the Materials and Methods. (A) Coomassie-stained SDS-PAGE and (B) immunoblots with antiserum against centrin and with monoclonal antibody 7A9. In (A) and (B), Lane 1, ICL, 40 µg; lane 2, ICL control pellet, 40 µg; lane 3, ICL EGTA pellet, 40 µg; lane 4, ICL control supernatant, 15 µg; lane 5, ICL EGTA supernatant, 15 µg; lane 6, PCBP-25, 3 µg; lane 7, PCBP-25ß, 5 µg. In (C), the 45Ca2+ blot overlay assay was performed as described in the Materials and Methods at 1.4 µM free Ca2+ with 20 µg of ICL EGTA supernatant. Coomassie-stained SDS-PAGE (lane 1) and autoradiograph of 45Ca2+ binding (lane 2). M, Mr of markers (x103).

View larger version (104K): [in a new window]   Fig. 7. EM localization of PCBP-25 and PCBP-25ß with gold-conjugated antibodies. (A-C) Using a polyclonal antibody against Chlamydomonas centrin, PCBP-25 was localised in the transition zone of the ciliary basal body (A) and in the ICL (B,C). (D-F) Using monoclonal antibody 7A9, PCBP-25ß was localised in the ICL (D,E) but not in the basal body (D,F). a, alveoli; ax, axoneme; bb, basal body; c, cilium; icl, infraciliary lattice; m, mitochondrion; pm, plasma membrane; tz, transition zone. Bar, 0.4 µM.

View larger version (38K): [in a new window]   Fig. 8. Phosphorylation of PCBP-25, PCBP-25ß and CaM by purified Ca2+-dependent protein kinases. CaPK-1 and CaPK-2 were purified by chromatography on phenyl-Sepharose and MonoQ. MonoQ-purified PCBP-25, PCBP-25ß and CaM were incubated with [-32P]ATP and either (A) CaPK-1 or (B) CaPK-2 (20 units each) for 30 minutes in the absence or presence of Ca2+ (13.5 µM free) according to the Materials and Methods. Lanes 1 and 2, 1 µg PCBP-25; lanes 3 and 4, 1 µg PCBP-25ß; lanes 5 and 6, 2 µg concentrated, desalted CaM. Arrows indicate autophosphorylation of CaPK-1 or CaPK-2 and phosphorylation of PCBP-25ß and CaM.

View larger version (57K): [in a new window]   Fig. 9. Phosphorylation of purified POK-2 and centrin. Overexpressed CaM (POK-2) and engineered centrin of Chlamydomonas were purified and prepared for phosphorylation by CaPK-1 and CaPK-2. Lanes 1 and 2, 2 µg POK-2; lanes 3 and 4, 1 µg centrin. Autophosphorylation of each kinase and the phosphorylation of POK-2 and centrin are indicated.

View larger version (49K): [in a new window]   Fig. 10. Time dependence and substrate dependence of CaM phosphorylation by CaPK-2. (A) Phosphorylation by CaPK-2 was performed with 2 µg CaM and 20 units of kinase in the presence of Ca2+. The same volume of reaction mixture was withdrawn at indicated time points, and the reaction was stopped by precipitating the protein with 15% TCA. (B) The amount of CaM for phosphorylation by 20 units of kinase was increased as indicated. The reaction was performed for 20 minutes at room temperature. Autophosphorylation of CaPK-2 (Mr 50,000) and CaM (Mr 17,000) phosphorylation are indicated by arrows. (C) The radioactivity incorporated into CaM was quantified from the dried gel using a Betagen detector system (Intelligenetics, Mountainview, CA) and plotted with time.

View larger version (42K): [in a new window]   Fig. 11. The effect of CaM antagonists on CaM phosphorylation. CaM antagonists melittin or calmidazolium were added to a reaction mixture of CaM phosphorylation by 20 units of CaPK-2 (in the presence of 13.5 µM free Ca2+; CaPK-2 is half-saturated at 0.2 µM free Ca2+). (A) Lane 1, control — no antagonist; lane 2, 0.5 µM; lane 3, 2 µM; lane 4, 5 µM; lane 5, 10 µM melittin. (B) Lane 1, 0.5 µM; lane 2, 2 µM; lane 3, 5 µM; lane 4, 10 µM calmidazolium.

View larger version (38K): [in a new window]   Fig. 12. Phosphorylation of several CaMs by CaPK-2. (A) Several Paramecium CaMs, including wild-type and behavioral mutants, and bovine CaM were tested for the substrate specificity of CaPK-2. (B) The relevant amino acid sequences are shown.

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