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Reengineering the signaling properties of a Src family kinase.

ABSTRACT: Src family kinases (SFKs) are modular signaling proteins possessing SH3, SH2, and tyrosine kinase domains. The SH3 and SH2 domains of SFKs have dual roles: they regulate the activity of the kinases, and they also target SFKs to their cellular substrates. We generated a series of novel SFKs by replacing the SH2 and SH3 domains of Hck with the syntrophin PDZ domain. In some constructs, the negative regulatory tyrosine in the C-terminal tail was also replaced with a PDZ ligand sequence. When expressed in mammalian cells, the substrate specificity of the PDZ-kinases was directed to a different group of proteins than wild-type Hck. The PDZ-kinases phosphorylate neuronal nitric oxide synthase (nNOS), a known binding partner of the syntrophin PDZ domain. We also introduced a PDZ ligand at the C-terminus of the adaptor protein Cas. PDZ-Hck kinases phosphorylate the engineered Cas protein in Cas(-/-) cells and restore the migration defect of these cells. A PDZ-kinase was also functional in rewiring MAPK signaling via an engineered ErbB2 construct containing a PDZ ligand sequence. Several of the PDZ-kinases show autoregulatory properties similar to natural SFKs. Thus, the PDZ-ligand interaction is able to functionally replace the normal SH2-pY527 interaction that regulates SFKs. Our data highlight the modularity and evolvability of signaling proteins.

SUBMITTER: Yadav SS

PROVIDER: S-EPMC2847435 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Reengineering the signaling properties of a Src family kinase.

Yadav Shalini S SS Yeh Brian J BJ Craddock Barbara P BP Lim Wendell A WA Miller W Todd WT

Biochemistry 20091101 46

Src family kinases (SFKs) are modular signaling proteins possessing SH3, SH2, and tyrosine kinase domains. The SH3 and SH2 domains of SFKs have dual roles: they regulate the activity of the kinases, and they also target SFKs to their cellular substrates. We generated a series of novel SFKs by replacing the SH2 and SH3 domains of Hck with the syntrophin PDZ domain. In some constructs, the negative regulatory tyrosine in the C-terminal tail was also replaced with a PDZ ligand sequence. When expres ...[more]

PMID: 19827767

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Project description:Reelin is a large glycoprotein with a dual role in the mammalian brain. It regulates the positioning and differentiation of postmitotic neurons during brain development and modulates neurotransmission and memory formation in the adult brain. Alterations in the Reelin signaling pathway have been described in different psychiatric disorders. Reelin mainly signals by binding to the lipoprotein receptors Vldlr and ApoER2, which induces tyrosine phosphorylation of the adaptor protein Dab1 mediated by Src family kinases (SFKs). In turn, phosphorylated Dab1 activates downstream signaling cascades, including PI3-kinase-dependent signaling. In this work, a mechanistic model based on ordinary differential equations was built to model early dynamics of the Reelin-mediated signaling cascade. Mechanistic models are frequently used to disentangle the highly complex mechanisms underlying cellular processes and obtain new biological insights. The model was calibrated on time-resolved data and a dose-response measurement of protein concentrations measured in cortical neurons treated with Reelin. It focusses on the interplay between Dab1 and SFKs with a special emphasis on the tyrosine phosphorylation of Dab1, and their role for the regulation of Reelin-induced signaling. Model selection was performed on different model structures and a comprehensive mechanistic model of the early Reelin signaling cascade is provided in this work. It emphasizes the importance of Reelin-induced lipoprotein receptor clustering for SFK-mediated Dab1 trans-phosphorylation and does not require co-receptors to describe the measured data. The model is freely available within the open-source framework Data2Dynamics (www.data2dynamics.org). It can be used to generate predictions that can be validated experimentally, and provides a platform for model extensions both to downstream targets such as transcription factors and interactions with other transmembrane proteins and neuronal signaling pathways.

Dataset Information

Reengineering the signaling properties of a Src family kinase.

Publications

Reengineering the signaling properties of a Src family kinase.

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