Thyroid hormone signaling

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We have continued to investigate the mechanism of PI3K stimulation by TRbeta and the consequences of PI3K signaling for the physiological effects of thyroid hormone. We have used a fluorescent PIP3 binding domain from the Akt protein kinase coupled with cyan and yellow fluorecent proteins to detect PIP3 production by fluorescence resonance energy transfer (FRET) in live cells in real time. Fret signals are blocked by inhibition of TRbeta with the nuclear receptor antagonist,1-850, and by wortmannin, an active site inhibitor of PI3K. PIP3 production is also blocked completely by 10 min preexposure to 100 nM TCDD, an environmental toxicant. We have used immunoprecipitation to show that TRbeta associates with the regulatory p85 subunit of PI3K in the absence of ligand but dissociates in the presence of thyroid hormone. We have also observed rapid thyroid hormone-dependent phosphorylation of the Akt protein kinase and recruitment of Rac to the plasma membrane confirming that thyroid hormone stimulates PI3K-dependent effectors. Finally we have discovered that the Src family kinase, Lyn, is part of the signaling complex and identified its binding site with mass spectrometry. Other proteins interact with p85 through two Src homology (SH2) domains which recognize phosphotyrosine. TRbeta but not TRalpha contains a consensus SH2-binding domain with high affinity for p85. Mutating the tyrosine in that consensus site to phenylalanine prevents reconstitution of Kv11.1 regulation by thyroid hormone in CHO cells but not activation of a heterologous transcription of a luciferase gene driven by a thyroid hormone receptor response element (TRE). A second tyrosine that is responsible for Lyn binding has been identified and is also required for PI3K stimulation. We have created a transgenic mouse line in which a Phe has been substituted for the Tyr. We have also discovered that dioxin (TCDD) blocks thyroid hormone signaling through PI3, and we have shown that the Ser/Thr protein phosphatase, PP5, which we previously identified as a Rac effector downstream of thyroid hormone signaling (Gentile et al 2006) protects neurons from the oxidative stress of exposure to amyloid beta. Thus thyroid hormone could contribute to human protectin from Alzheimer's disease.