Phosphorylation of Numb family proteins. Possible involvement of Ca2+/calmodulin-dependent protein kinases.
To search for the substrates of Ca2+/calmodulin-dependent protein kinase I (CaM-KI), we performed affinity chromatography purification using either the unphosphorylated or phosphorylated (at Thr177) GST-fused CaM-KI catalytic domain (residues 1-293, K49E) as the affinity ligand. Proteomic analysis was then carried out to identify the interacting proteins. In addition to the detection of two known CaM-KI substrates (CREB and synapsin I), we identified two Numb family proteins (Numb and Numbl) from rat tissues. These proteins were unphosphorylated and were bound only to the Thr177-phosphorylated CaM-KI catalytic domain. This finding is consistent with the results demonstrating that Numb and Numbl were efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser264 in Numb and Ser304 in Numbl) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phospho-Numb/Numbl antibody, we observed the phosphorylation of Numb family proteins in various rat tissue extracts, and we also detected the ionomycin-induced phosphorylation of endogenous Numb at Ser264 in COS-7 cells. The present results revealed that the Numb family proteins are phosphorylated in vivo as well as in vitro. Furthermore, we found that the recruitment of 14-3-3 proteins was the functional consequence of the phosphorylation of the Numb family proteins. Interaction of 14-3-3 protein with phosphorylated Numbl-blocked dephosphorylation of Ser304. Taken together, these results indicate that the Numb family proteins may be intracellular targets for CaM-Ks, and they may also be regulated by phosphorylation-dependent interaction with 14-3-3 protein.
Tokumitsu
Hiroshi
H
Department of Signal Transduction Sciences, Faculty of Medicine, Kagawa University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan. tokumit@med.kagawa-u.ac.jp
Hatano
Naoya
N
Inuzuka
Hiroyuki
H
Sueyoshi
Yuka
Y
Yokokura
Shigeyuki
S
Ichimura
Tohru
T
Nozaki
Naohito
N
Kobayashi
Ryoji
R
D048948:14-3-3 Proteins / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry*; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D029721:Drosophila Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D007605:Juvenile Hormones / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D016704:Synapsins / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D013997:Time Factors; D014018:Tissue Distribution; D014162:Transfection
D048948:14-3-3 Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D018076:DNA, Complementary; D029721:Drosophila Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007605:Juvenile Hormones; D009419:Nerve Tissue Proteins; C106292:Numbl protein, mouse; C506057:Numbl protein, rat; D016704:Synapsins; C060606:numb protein, Drosophila; D013912:Threonine; D012694:Serine; D015759:Ionomycin; D005982:Glutathione Transferase; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517598:Camk1 protein, mouse; C517599:Camk1 protein, rat; C517681:Pnck protein, mouse; C517682:Pnck protein, rat; D002118:Calcium