Shigeyuki Nada

Identification of PSD-93 as a Substrate for the Src Family Tyrosine Kinase Fyn

In order to study the role of tyrosine kinase signaling in the post-synaptic density (PSD), tyrosine-phosphorylated proteins associated with the PSD-95/NMDA receptor complex were analyzed. The NMDA receptor complex from the mouse brain was successfully solubilized with deoxycholate and immunopurified with anti-PSD-95 or anti-phosphotyrosine antibody. Immunoblot analyses revealed that the predominantly tyrosine-phosphorylated proteins in the NMDA receptor complex are the NR2A/B subunits and a novel 120 kDa protein. Purification and microsequencing analysis showed that the 120 kDa protein is mouse PSD-93/Chapsyn-110. Recombinant PSD-93 was phosphorylated by Fyn in vitro, and Tyr-384 was identified as a major phosphorylation site. Tyrosine phosphorylation of PSD-93 was greatly reduced in brain tissue from Fyn-deficient mice compared with wild-type mice. Furthermore, an N-terminal palmitoylation signal of PSD-93 was found to be essential for its anchoring to the membrane, where Fyn is also localized. In COS7 cells, exogenously expressed PSD-93 was phosphorylated, dependent on its membrane localization. In addition, tyrosine-phosphorylated PSD-93 was able to bind to Csk, a negative regulator of Src family kinases, in vitro as well as in a brain lysate. These results suggest that PSD-93 serves as a membrane-anchored substrate of Fyn and plays a role in the regulation of Fyn-mediated modification of NMDA receptor function.

Roles of C-terminal Src kinase in the initiation and the termination of the high affinity IgE receptor-mediated signaling.

As an attempt to analyze the roles of C-terminal Src kinase (Csk) in the high affinity IgE receptor (FcεRI)-mediated signaling, we overexpressed Csk, a membrane-targeted form of Csk (mCsk), and a kinase-defective, membrane-targeted form of Csk (mCsk(−)) in rat basophil leukemia (RBL) 2H3 cells. Specific activity of Lyn at the basal state was decreased in Csk-expressing cells, and further decreased in mCsk-expressing cells. In mCsk(−)-expressing cells, basal specific activity of Lyn was increased, thereby indicating that mCsk(−) functioned as a dominant negative molecule. The onset of FcεRI-mediated Lyn activation was delayed in Csk-expressing cells, and further delayed in mCsk-expressing cells. In mCsk(−)-expressing cells, Lyn activation was rapid and quite long lasting. These findings indicate (i) Csk negatively regulates rapid FcεRI/Lyn coupling, and (ii) Csk activity is potentially required for its termination. The onsets of the series of events including tyrosyl phosphorylation of Syk, mitogen-activated protein (MAP) kinase activation, elevation of intracellular calcium concentration ([Ca2+] i ), and histamine release were all stepwisely delayed in Csk-expressing cells and in mCsk-expressing cells. The durations of Syk phosphorylation and MAP kinase activation also closely correlated with those of Lyn activation, but [Ca2+] i elevation and histamine release followed different temporal patterns: the delayed responses in Csk-expressing cells and in mCsk-expressing cells led to sustained [Ca2+] i oscillation and histamine release, while the prompt responses in parent cells and mCsk(−)-expressing cells rapidly subsided. These findings provide further evidence that the initiations of the FcεRI-mediated signals are upstreamly regulated by Src family protein tyrosine kinases and revealed that their terminations are regulated by Lyn-dependent (Syk and MAP kinase) and -independent ([Ca2+] i elevation and histamine release) mechanisms.