Sustained Adrenergic Stimulation Is Required for the Nuclear Retention of TORC1 in Male Rat Pinealocytes

Abstract

The process involved in relocation of the coactivator, transducer of regulated cAMP-regulated element-binding protein (TORC) to the cytoplasm, unlike its activation, is not well understood. Using cultured pineal cells prepared from male rats, we found that although both α- and β-adrenergic stimulation could cause TORC1 dephosphorylation, only α-adrenergic stimulation was effective in the norepinephrine (NE)-mediated translocation of TORC1 into the nucleus. In contrast, blockade of either the α- or the β-adrenergic receptor after NE stimulation was effective in causing the rephosphorylation and rapid relocation of TORC1 into the cytoplasm. Studies with phosphoprotein phosphatase (PP) inhibitors indicated that although both PP2A and PP2B could dephosphorylate TORC1, only PP2B could cause translocation into the nucleus. However, after NE stimulation, treatment with either PP2A or PP2B inhibitors could cause the rephosphorylation and cytoplasmic relocation of TORC1. These results indicate a requirement of continuous activation of both α- and β-adrenergic receptors as well as PP2A and PP2B activities for the nuclear retention of TORC1 during NE stimulation. Knockdown of salt-inducible kinase 1 (SIK1) had no effect on the phosphorylation or localization of TORC1. Although overexpressing SIK1 could induce TORC1 phosphorylation in the nucleus, it did not reduce TORC1 level in the nucleus, indicating that SIK1-mediated TORC1 phosphorylation may not be sufficient for its relocation into the cytoplasm. Together, these results demonstrate that, in the rat pineal gland, different mechanisms are involved in regulating the nuclear entry and exit of TORC1 and that the SIK1-mediated phosphorylation of TORC1 may not lead to its nuclear exit.