SLAM-associated Protein as a Potential Negative Regulator in Trk Signaling

Abstract

Neurotrophin signaling plays important roles in regulating the survival, differentiation, and maintenance of neurons in the nervous system. Binding of neurotrophins to their cognate receptors Trks induces transactivation and phosphorylation of the receptor at several tyrosine residues. These phosphorylated tyrosine residues then serve as crucial docking sites for adaptor proteins containing a Src homology 2 or phosphotyrosine binding domain, which upon association with the receptor initiates multiple signaling events to mediate the action of neurotrophins. Here we report the identification of a Src homology 2 domain-containing molecule, SLAM-associated protein (SAP), as an interacting protein of TrkB in a yeast two-hybrid screen. SAP was initially identified as an adaptor molecule in SLAM family receptor signaling for regulating interferon-␥ secretion. In the current study, we found that SAP interacted with TrkA, TrkB, and TrkC receptors in vitro and in vivo. Binding of SAP required Trk receptor activation and phosphorylation at the tyrosine 674 residue, which is located in the activation loop of the kinase domain. Overexpression of SAP with Trk attenuated tyrosine phosphorylation of the receptors and reduced the binding of SH2B and Shc to TrkB. Moreover, overexpression of SAP in PC12 cells suppressed the nerve growth factor-dependent activation of extracellular signal-regulated kinases 1/2 and phospholipase C␥, in addition to inhibiting neurite outgrowth. In summary, our findings demonstrated that SAP may serve as a negative regulator of Trk receptor activation and downstream signaling.

Cdk5 Is Involved in BDNF-Stimulated Dendritic Growth in Hippocampal Neurons

“…HA-tagged and GST-tagged Rac1, Cdc42, and RhoA constructs were gifts from Yung-Hou Wong (Hong Kong University of Science and Technology, Hong Kong). The expression vectors of TrkA, TrkB, and TrkC were constructed as described [33]. Three TrkB mutants lacking the potential Cdk5 phosphorylation sites were constructed by mutating Ser478 (TrkB M1), Thr489 (TrkB M2), or both Ser478 and Thr489 (TrkB DM) to alanine using the overlapping PCR technique, followed by subcloning into pcDNA3.…”

“…Rat cortical and hippocampal neuron cultures were prepared as previously described [33,34]. Subsequent to digestion with 0.25% trypsin in Hank's Balanced Salt Solution without Ca 2+ and Mg 2+ at 37 °C for 5 min, the reaction was stopped by 2.5% heat-inactivated horse serum.…”

Negative Regulation of Receptor Tyrosine Kinase (RTK) Signaling: A Developing Field

“…Binding of SAP requires Trk receptor activation and phosphorylation of the tyrosine 674, which is located in the activation loop of the kinase domain. Moreover, overexpression of SAP attenuates tyrosine phosphorylation of Trk receptors and suppress NGF-induced neurite outgrowth (Lo et al 2005). …”

The role of the SLAM-SAP signaling pathway in the modulation of CD4+ T cell responses

“…In addition, SAP promotes the recruitment and enzymatic activation of protein tyrosine kinase Fyn T, inducing the phosphorylation of the cytoplasmic domain of SLAM and allowing the propagation of events of the signaling cascade (20,42). In neural cells, SAP exerts its role through the recruitment of protein kinases related to tropomyosin (39).…”