ABSTRACT
Kalirin-7 (Kal7), a multifunctional Rho GDP/GTP exchange factor (GEF) for Rac1 and RhoG, is embedded in the postsynaptic density at excitatory synapses, where it participates in the formation and maintenance of dendritic spines. Kal7 has been implicated in long-term potentiation, fear memories, and addiction-like behaviors. Using liquid chromatography and tandem mass spectroscopy, we identified sites phosphorylated by six PSD-localized kinases implicated in synaptic plasticity and behavior, sites phosphorylated when myc-Kal7 was expressed in non-neuronal cells and sites phosphorylated in mouse brain Kal7. A site in the Sec14p domain phosphorylated by calcium/calmodulin dependent protein kinase II, protein kinase A and protein kinase C, was phosphorylated in mouse brain but not in non-neuronal cells. Phosphorylation in the spectrin-like repeat region was more extensive in mouse brain than in non-neuronal cells, with a total of 20 sites identified. Sites in the pleckstrin homology domain and in the linker region connecting the GEF domain to the PDZ binding motif were heavily phosphorylated in both non-neuronal cells and in mouse brain and affected GEF activity. We postulate that the kinase convergence and divergence observed in Kal7 identify it as a key player in integration of the multiple inputs that regulate synaptic structure and function.
Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Post-Synaptic Density/metabolism , Recombinant Fusion Proteins/metabolism , Synaptic Transmission , Amino Acid Motifs , Amino Acid Sequence , Animals , Brain/metabolism , Catalytic Domain , Cell Line , Guanine Nucleotide Exchange Factors/chemistry , Guanine Nucleotide Exchange Factors/genetics , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation, Missense , Peptide Fragments/chemistry , Phosphorylation , Protein Serine-Threonine Kinases/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Tandem Mass Spectrometry , Threonine/chemistry , Threonine/genetics , Threonine/metabolismABSTRACT
Isotope-coded affinity tags (ICAT) is a labeling technique that provides insights into quantitative molecular changes. In this paper, we propose a new protocol to identify and analyze ICAT labeled peak pairs in high-resolution LC-MS data. Our major contributions are: (1) we use isotope distance constraint, ICAT distance constraint, and LC-span constraint to identify ICAT labeled peak pairs and (2) we propose to trigger tandem MS/MS scanning based on the ratio estimation value of identified ICAT peak pairs instead of the peak intensity values. Compared with current approaches that choose peaks with high intensity values for tandem MS/MS scanning, the new protocol can improve the scanning efficiency and accuracy.