Jun Qin

Preferential phosphorylation of R-domain Serine 768 dampens activation of CFTR channels by PKA.

CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTRs two cytoplasmic nucleotide binding domains, but only after several serines in CFTRs regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (Po) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted Po-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals.

Modulation of GT-1 DNA-binding activity by calcium-dependent phosphorylation

The analysis of pea rbcS-3A promoter sequence showed that BoxII was necessary for the control of rbcS-3A gene expression by light. GT-1, a DNA-binding protein that interacts with BoxII in vitro, is a good candidate for being a light-modulated molecular switch controlling gene expression. However, the relationship between GT-1 activity and light-responsive gene activation still remains hypothetical. Because no marked dexa0novo synthesis was detected after light treatment, light may induce post-translational modifications of GT-1 such as phosphorylation or dephosphorylation. Here, we show that recombinant GT-1 (hGT-1) of Arabidopsis can be phosphorylated by various mammalian kinase activities inxa0vitro. Whereas phosphorylation by casein kinasexa0II had no apparent effect on hGT-1 DNA binding, phosphorylation by calcium/calmodulin kinasexa0II (CaMKII) increased the binding activity 10–20-fold. Mass spectrometry analyses of the phosphorylated hGT-1 showed that amongst the 6 potential phosphorylatable residues (T86, T133, S175, T179, S198 and T278), only T133 and S198 are heavily modified. Analyses of mutants altered at T86, T133, S175, T179, S198 and T278 demonstrated that phosphorylation of T133 can account for most of the stimulation of DNA-binding activity by CaMKII, indicating that this residue plays an important role in hGT-1/BoxII interaction. We further showed that nuclear GT-1 DNA-binding activity to BoxII was reduced by treatment with calf intestine phosphatase in extracts prepared from light-grown plants but not from etiolated plants. Taken together, our results suggest that GT-1 may act as a molecular switch modulated by calcium-dependent phosphorylation and dephosphorylation in response to light signals.

Collision-induced dissociation of singly charged peptide ions in a matrix-assisted laser desorption ionization ion trap mass spectrometer

Abstract We document the systematics of collision-induced dissociation of singly charged peptide ions in a matrix-assisted laser desorption/ionization (MALDI) ion trap mass spectrometer. We show that singly charged peptide ions with m/z ratios extending to 3500 can be effectively fragmented using a newly devised excitation scheme, termed red shifted off-resonance large amplitude excitation (RSORLAE), and classify the dominant features of the resulting collision-induced dissociation spectra as follows. (1) Peptides ions with a m/z value of m/z

Linker chains of the gigantic hemoglobin of the earthworm Lumbricus terrestris: Primary structures of linkers L2, L3, and L4 and analysis of the connectivity of the disulfide bonds in linker L1†‡

The extracellular hemoglobin (Hb) of the earthworm, Lumbricus terrestris, has four major kinds of globin chains: a, b, c, and d, present in equimolar proportions, and additional non‐heme, non‐globin scaffolding chains called linkers that are required for the calcium‐dependent assembly of the full‐sized molecule. The amino acid sequences of all four of the globin chains and one of the linkers (L1) have previously been determined. The amino acid sequences via cDNA of each of the three remaining linkers, L2, L3, and L4, have been determined so that the sequences of all constituent polypeptides of the hemoglobin are now known. Each linker has a highly conserved cysteine‐rich segment of ∼ 40 residues that is homologous with the seven ligand‐binding repeats of the human low‐density lipoprotein receptor (LDLR). Analysis of linker L1 shows that the connectivity of the three disulfide bonds is exactly the same as in the LDLR ligand‐binding repeats. The presence of a calcium‐binding site comprising one glutamyl and three aspartyl residues in both the LDLR repeats and in the linkers supports the suggestion that calcium is required for the folding and disulfide connectivity of the linkers as in the LDLR repeats. Linker L2 is markedly heterogeneous and contains unusual glycine‐rich sequences near the NH2‐terminus and a polar zipper‐like sequence with imperfect repeats of Asp‐Asp‐His at the carboxyl terminus. Similar Asp‐Asp‐His repeats have been found in a protein homologous to superoxide dismutase in the hemolymph of certain mussels. These repeats may function as metal‐binding sites. Proteins 2006.