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Pelle kinase is activated by autophosphorylation during Toll signaling in Drosophila

Department of Biological Sciences, Columbia University, New York, NY 10027, USA

View larger version (45K): [in a new window]   Fig. 1. Signal-dependent phosphorylation of Pelle during early embryogenesis. (A) Pelle is transiently modified in early embryos. Whole embryo extract was prepared from wild-type embryos of different stages (see Materials and Methods), and equivalent amounts of extract were subjected to SDS-PAGE. Pelle was detected by anti-Pelle immunoblotting (top). Anti-Toll immunoblotting was used as control (bottom). Lanes 1-6, embryo extracts were from 1- to 6-hour-old embryos as indicated. (B) Pelle modification is enhanced in gain-of-function Toll mutant embryos. Wild-type or mutant embryos with an activated Toll allele (Toll10b) were collected and analyzed as in A. The top panel is an anti-Pelle blot, and the bottom panel is a blot with anti-Toll antiserum. Lanes 1-5, wild-type embryos between 0 and 5 hours after egg laying; lanes 6-10, Toll10b embryos between 0 and 5 hours after egg laying. (C) Pelle modification is decreased in loss-of-function Toll mutant embryos. Wild-type, Toll10b, gastrulation defective null mutant (Gd–/–), weak loss-of-function Toll mutant (Tollr444) and Toll knockout (9QRX) embryos were collected 2-4 hours after egg laying and analyzed by SDS-PAGE and western blot as in A. (D) Pelle is phosphorylated in Toll10b embryos. For dephosphorylation by endogenous phosphatases, Toll10b whole embryo extract from the second to fourth hours after egg laying (lane 1) was incubated at 30°C with or without NaF (lane 2, 3) for 1 hour. The same extract was also incubated with immobilized calf intestine phosphatase (CIP-beads), with or without NaF (lane 4, 5), at 37°C for 1 hour. Pelle protein was analyzed by SDS-PAGE followed by anti-Pelle immunoblotting.

View larger version (31K): [in a new window]   Fig. 2. Concentration-dependent Pelle phosphorylation in SL2 cells. (A) Pelle is autophosphorylated in SL2 cells when overexpressed. Increasing amounts of Pelle or PelleK240R expression vectors were transfected into SL2 cells. After 48 hours, cells were lysed, and subjected to anti-Pelle (A, top) or anti-Dorsal (A, bottom) immunoblots. The amount of cell lysate used was normalized by ß-Gal assays. Lane 1, vector; lanes 2-5: 0.03, 0.1, 0.3 or 1 µg of PelleK240R DNA was transfected. Lanes 6-9: 0.03, 0.1, 0.3 and 1 µg of Pelle DNA was transfected. (B) Pelle autophosphorylation occurs concomitantly with Dorsal activation. Cell lysates from the same transfections as above were used for CAT assays. Dorsal activation was quantitated by measuring expression from the co-transfected Dorsal-responsive CAT reporter. CAT activity is represented by the ratio of CAT/ß-Gal (see Materials and Methods).

View larger version (22K): [in a new window]   Fig. 3. Pelle is autophosphorylated when co-transfected with constitutive active Toll10b. (A) The indicated amounts of DNA were transfected into SL2 cells, and then analyzed as Fig. 2. Lane 1, 0.3 µg of Toll10b; lane 2, 0.1 µg of Pelle; lane 3, 0.3 µg Toll10b plus 0.1 µg Pelle; lane 4, 0.1 µg PelleK240R; and lane 5, 0.3 µg Toll10b plus 0.1 µg of PelleK240R. Western blots of transfected cell lysates: top, anti-Pelle blot; bottom, anti-Dorsal blot. (B) CAT assays for the same transfections were performed as in Fig. 2.

View larger version (35K): [in a new window]   Fig. 4. Autophosphorylation is necessary for Pelle kinase activity. (A) Both autophosphorylated HisPelle (lane 2) and dephosphorylated CIP-Pelle (lane 1) were expressed in and purified from E. coli (see Materials and Methods), and subjected to SDS-PAGE followed by anti-Pelle western blotting. (B) Different forms of Pelle were incubated in kinase buffer containing [-32P]ATP, with or without HisTubeN, at 30°C for 30 minutes. Reactions were terminated by TCA precipitation, and precipitates were washed and subjected to SDS-PAGE. Phosphate incorporation was analyzed by autoradiography. Top: lane 1, 100 ng of HisTubeN; lane 2, 200 ng of CIP-Pelle; lane 3, 100 ng of HisTubeN plus 200 ng of CIP-Pelle; lane 4, 200 ng of HisPelle; and lane 5, 100 ng of HisTubeN and 200 ng of HisPelle. There were equivalent amounts of Pelle (anti-Pelle blot) and HisTubeN (anti-His blot) in lanes 3 and 5 (bottom).

View larger version (30K): [in a new window]   Fig. 5. CIP-Pelle can be re-autophosphorylated and activated in a concentration-dependent manner in vitro. (A) CIP-Pelle was first incubated in kinase buffer at the indicated concentrations, at 30°C for 1 hour. Identical amounts of pre-incubated Pelle were then removed and incubated in kinase buffer at an identical concentration (5 µg/ml) with 5 µg/ml of HisTubeN at 30°C for 30 minutes. Reaction mixtures were adjusted to contain identical concentrations of buffer components and ATP. Identical aliquots were loaded onto an SDS gel and analyzed as in Fig. 4B. The different CIP-Pelle concentrations during preincubation are indicated: lanes 1, 2 and 3 contained CIP-Pelle at 25, 50 and 100 µg/ml, respectively. (B) CIP-Pelle and HisTubeN in the final reaction mixtures analyzed in Fig. 5A were detected by western blotting with anti-Pelle and anti-His antibodies, respectively. Two exposures of the anti-Pelle blot are shown and the positions of distinct Pelle isoforms are indicated.

View larger version (27K): [in a new window]   Fig. 6. Pelle is transphosphorylated and capable of self-association. (A) GST-PelleK240R is phosphorylated by CIP-Pelle. GST-PelleK240R and/or CIP-Pelle was incubated in kinase buffer containing [-32P]ATP as in Fig. 4. Proteins were analyzed by SDS-PAGE as in Fig. 5A. Lane 1, 250 ng CIP-Pelle; lane 2, 250 ng CIP-Pelle and 500 ng GST-PelleK240R; and lane 3, 500 ng GST-PelleK240R. (B) Pelle self-association. Purified GST or GST-Pelle (2 µg) was immobilized on glutathione beads, and incubated with 1 µg of purified CIP-Pelle. Protein complexes were eluted with 20 mM glutathione (reduced form) and analyzed by SDS-PAGE followed by anti-Pelle immunoblot. Lane 1, 20% of CIP-Pelle used in binding assays (input); lanes 2 and 3, GST or GST-PelleK240R, respectively, incubated with CIP-Pelle.

View larger version (25K): [in a new window]   Fig. 7. Molecular model for early signaling and Pelle activation in the Toll pathway. A membrane-localized Toll/Tube/Pelle complex forms, perhaps transiently, before signaling (Toll TIR and inhibitory domains and Pelle regulatory and catalytic domains are indicated). Upon binding of the Toll ligand Spätzle, a high local Pelle concentration, induced by the resulting Toll dimerization (broken lines), is created, and this leads to Pelle transphosphorylation (arrows) and activation. Activated Pelle then phosphorylates the Toll Tir domain and Tube (curved arrows), Pelle (and Tube) are released from Toll, and Pelle phosphorylates downstream targets. See text for additional details.

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