Phosphorylation of Syk in the linker B region modulates downstream signaling to the transcription factor NFAT

Victoria Aileen Martin, Purdue University

Abstract

Misregulation of B lymphocyte responses to foreign antigens is linked to a variety of immune diseases, including allergies, autoimmunities, and cancer. Understanding the molecular mechanisms that regulate such responses is therefore critical to the design of therapeutics for these disorders. Ligation of the B cell antigen receptor leads to the phosphorylation and activation of Syk kinase, a critical initiation step for signaling to the activation of transcription factors, such as NFAT, that modulate cellular responses. It has been shown that the phosphorylation of particular tyrosine residues on Syk varies with different cell stimuli and can regulate Syk binding partner selection. In this study, I investigated the role of phosphorylation of three sites on Syk in the modulation of downstream signaling pathways and the activation of NFAT. First, I examined two closely spaced tyrosines, Y342 and Y346, in the interaction between Syk and the proto-oncogene Vav1. I found that the stoichiometry of phosphorylation on these two residues altered the Syk-dependent phosphorylation of Vav1 and downstream activation of NFAT. NMR spectroscopy revealed that this regulation was dependent on conserved lysine residues within a secondary binding pocket of the Vav1 SH2 domain that interacted with pY346. There was a strong correlation between in vitro Syk-Vav1 binding affinity and both Vav1 phosphorylation and downstream NFAT activation with different phosphorylation patterns on Y342 and Y346, indicating the importance of Syk phosphorylation at these sites for regulation of Vav1 signaling to NFAT. Furthermore, phosphopeptide pulldown analyses using sequences from a kinase unrelated to Syk suggested that the regulation of kinase binding to SH2 domain-containing substrates by the phosphorylation of two closely spaced tyrosines may be a widespread means of modulating signaling. Next I investigated signaling downstream of another Syk phosphorylation site, Y317. Signaling through Syk, with or without Y317 phosphorylation, to the activation of NFAT was differentially inhibited by the PI3K inhibitor wortmannin, but PI3K itself was activated in both cases. Further investigation revealed that both positive and negative NFAT regulators were activated downstream of PI3K, and that, contrary to what is predicted by the current literature, Akt2 inhibited NFAT activity. A detailed analysis of the signaling pathway indicated that Akt2, but not Akt1, inhibited the prolonged calcium influx required for NFAT activation selectively over the initial influx that activates NFkB. Inhibition of the interaction between Bcl-2 and IP3R1 rescued NFAT activation from Akt2 inhibition. I hypothesize that Akt2 inhibits NFAT through phosphorylation of a Bcl-2 sequestering protein, likely the known Akt substrate BAD, releasing Bcl-2 to inhibit IP3R1 function and limit calcium efflux. Furthermore, experiments using Akt mutants suggested that localization may be important for the isoform differences I observed. Together, these studies elucidate two mechanisms by which Syk phosphorylation at specific tyrosines regulates downstream signaling and transcription factor activation.

Degree

Ph.D.

Advisors

Geahlen, Purdue University.

Subject Area

Molecular biology

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