Salt‐inducible kinase‐1 represses cAMP response element‐binding protein activity both in the nucleus and in the cytoplasm

Abstract

Salt-inducible kinase-1 (SIK1) is phosphorylated at Ser577 by protein kinase A in adrenocorticotropic hormone-stimulated Y1 cells, and the phospho-SIK1 translocates from the nucleus to the cytoplasm. The phospho-SIK1 is dephosphorylated in the cytoplasm and re-enters the nucleus several hours later. By using green-fluorescent protein-tagged SIK1 fragments, we found that a peptide region (586-612) was responsible for the nuclear localization of SIK1. The region was named the ÔRK-rich regionÕ because of its Arg-and Lys-rich nature. SIK1s mutated in the RK-rich region were localized mainly in the cytoplasm. Because SIK1 represses cAMP-response element (CRE)-mediated transcription of steroidogenic genes, the mutants were examined for their effect on transcription. To our surprise, the cytoplasmic mutants strongly repressed the CRE-binding protein (CREB) activity, the extent of repression being similar to that of SIK1(S577A), a mutant localized exclusively in the nucleus. Several chimeras were constructed from SIK1 and from its isoform SIK2, which was localized mainly in the cytoplasm, and they were examined for intracellular localization as well as CREB-repression activity. A SIK1-derived chimera, where the RK-rich region had been replaced with the corresponding region of SIK2, was found in the cytoplasm, its CREB-modulating activity being similar to that of wild-type SIK1. On the other hand, a SIK2-derived chimera with the RK-rich region of SIK1 was localized in both the nucleus and the cytoplasm, and had a CREBrepressing activity similar to that of the wild-type SIK2. Green fluorescent protein-fused transducer of regulated CREB activity 2 (TORC2), a CREB-specific co-activator, was localized in the cytoplasm and nucleus of Y1 cells, and, after treatment with adrenocorticotropic hormone, cytoplasmic TORC2 entered the nucleus, activating CREB. The SIK1 mutants, having a strong CRE-repressing activity, completely inhibited the adrenocorticotropic hormoneinduced nuclear entry of green fluorescent protein-fused TORC2. This suggests that SIK1 may regulate the intracellular movement of TORC2, and as a result modulates the CREB-dependent transcription activity. Together, these results indicate that the RK-rich region of SIK1 is important for determining the nuclear localization and attenuating CREB-repressing activity, but the degree of the nuclear localization of SIK1 itself does not necessarily reflect the degree of SIK1-mediated CREB repression.

PGE2 Induces Macrophage IL-10 Production and a Regulatory-like Phenotype via a Protein Kinase A–SIK–CRTC3 Pathway

“…Our data would indicate that Ser 343 is the critical site on SIK2 for regulation via PKA in macrophages. Ser 358 and Ser 587 in SIK2, or the corresponding Ser 577 site in SIK1, have previously been suggested as PKA sites in other cell types (55)(56)(57). Although we detected these sites in macrophages (Table II), we did not observe any increase in their phosphorylation in response to PGE 2 , indicating that they are unlikely to be involved in our system.…”

Salt-inducible Kinase 3 Signaling Is Important for the Gluconeogenic Programs in Mouse Hepatocytes

“…Although multiple SIK substrates have been identified based on information from the SIK phosphorylation motifs, LX(R/K)(S/ T)XpSXXXL (5), only two types of substrates, CRTC (a coactivator for the cAMP response element (CRE)-binding protein (CREB)) and class IIa histone deacetylase (HDAC) (6), have been confirmed by independent research groups. Phosphorylation of these two substrates by SIKs results in a loss of their transcriptional regulatory activities by inducing nuclear export (5,7).…”

“…When PKA activates CREB by phosphorylation, it also counters SIK actions by phosphorylating the C-terminal regulatory domain of SIK, which in turn promotes the dephosphorylation rate of CRTC, enhances nuclear accumulation of this coactivator, and increases CREB-dependent gene expression (5,7,9). Conversely, when PKA activity wanes, the reactivated SIK terminates CREB activity by inactivating CRTC (5,9).…”

AMPK-dependent Repression of Hepatic Gluconeogenesis via Disruption of CREB·CRTC2 Complex by Orphan Nuclear Receptor Small Heterodimer Partner

“…CREB coactivator CRTC2 significantly contributes to the CRE-dependent transcriptional activation of hepatic gluconeogenesis (11). Under feeding or in the presence of insulin, CRTC2 is located in the cytoplasm via its phosphorylation at Ser 171 by members of the AMPK family of Ser/Thr kinase, including AMPK and SIK1 (salt-inducible kinase 1) (11,32). Fasting triggers activation of cAMP-dependent protein kinase (PKA) to promote dephosphorylation and nuclear entry of CRTC2, which results in the increased occupancy of CRTC2 over promoters of PEPCK, G6Pase, or PGC-1␣ and activation of the entire gluconeogenic program in mouse liver or in rat primary hepatocytes (33,34).…”