Phosphorylation-dependent interaction of kinesin light chain 2 and the 14-3-3 protein.
The protein 14-3-3 is a key regulator in a cell signaling pathway mediated by protein phosphorylation. To identify the cellular targets of this protein systematically, we have employed a proteomic approach: protein components pulled down from PC12 cells stably expressing a myc-tagged 14-3-3eta isoform were analyzed by means of SDS-PAGE and mass spectrometry. This procedure allowed us to identify more than 30 proteins that include various known and unknown targets of the 14-3-3 protein. Among them are several proteins in the membrane traffic pathway, such as the heavy and light chains (KHC/KIF5B and KLC2) of conventional kinesin, a heterotetrameric mechanochemical motor involved in the ATP-dependent movement of vesicles and organelles along microtubules. Subsequent analysis showed that 14-3-3 directly binds to kinesin heterodimers through interaction with KLC2 and that this interaction is dependent on the phosphorylation of KLC2. Studies on the interaction between 14-3-3 and KLC2 variants expressed in cultured cells coupled with mass spectrometric analysis proved that Ser575 is the site of phosphorylation in KLC2 that is responsible for the in vivo interaction with the 14-3-3 protein. These data add KLC2 to the growing list of 14-3-3 targets, and suggest a role of 14-3-3 in the phosphorylation-regulated cellular transport of vesicles and organelles.
Ichimura
Tohru
T
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Hachioji-shi, Tokyo 192-0397, Japan. ichimura@mail.comp.metro-u.ac.jp
Wakamiya-Tsuruta
Akiko
A
Itagaki
Chiharu
C
Taoka
Masato
M
Hayano
Toshiya
T
Natsume
Tohru
T
Isobe
Toshiaki
T
D048948:14-3-3 Proteins; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D017124:Conserved Sequence; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D051381:Rats; D012694:Serine / Q000235:genetics; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D048948:14-3-3 Proteins; D007527:Isoenzymes; C486567:KIF15 protein, human; C000715692:Klc2 protein, rat; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; C482984:YWHAH protein, human; D010768:Phosphoserine; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D016547:Kinesins; D000409:Alanine