Silencing the constitutive active transcription factor CREB by the LKB1‐SIK signaling cascade

Abstract

Cyclic AMP-responsive element (CRE)-binding protein (CREB) is a transcription factor that plays an important role in numerous physiological events, such as cell proliferation, survival, tumorigenesis, glucose metabolism and memory, in a phosphorylation-dependent manner [1,2] Cyclic AMP responsive element (CRE)-binding protein (CREB) is known to activate transcription when its Ser133 is phosphorylated. Two independent investigations have suggested the presence of Ser133-independent activation. One study identified a kinase, salt-inducible kinase (SIK), which repressed CREB; the other isolated a novel CREB-specific coactivator, transducer of regulated CREB activity (TORC), which upregulated CREB activity. These two opposing signals are connected by the fact that SIK phosphorylates TORC and induces its nuclear export. Because LKB1 has been reported to be an upstream kinase of SIK, we used LKB1-defective HeLa cells to further elucidate TORC-dependent CREB activation. In the absence of LKB1, SIK was unable to phosphorylate TORC, which led to constitutive activation of CRE activity. Overexpression of LKB1 in HeLa cells improved the CRE-dependent transcription in a regulated manner. The inactivation of kinase cascades by 10 nm staurosporine in LKB1-positive HEK293 cells also induced unregulated, constitutively activated, CRE activity. Treatment with staurosporine completely inhibited SIK kinase activity without any significant effect on the phosphorylation level at the LKB1-phosphorylatable site in SIK or the activity of AMPK, another target of LKB1. Constitutive activation of CREB in LKB1-defective cells or in staurosporine-treated cells was not accompanied by CREB phosphorylation at Ser133. The results suggest that LKB1 and its downstream SIK play an important role in silencing CREB activity via the phosphorylation of TORC, and such silencing may be indispensable for the regulated activation of CREB.Abbreviations A-loop, activation loop; AMPK, AMP-activated protein kinase; bZIP, basic leucine zipper domain; CRE, cAMP-response element; CREB, CRE-binding protein; DAPI, 4¢,6-diamidino-2-phenylindole; GFP, green fluorescent protein; GST, glutathione-S-transferase; HA, hemagglutinin; KID, kinase-inducible domain; moi, multiplicities of infection; PKA, protein kinase A; RT, reverse transcription; SIK, salt-inducible kinase; TORC, transducer of regulated CREB activity.

Enhanced activity of the CREB co-activator Crtc1 in LKB1 null lung cancer

“…As LKB1 has been identified as a key regulator for Crtc2 transcriptional activity (Shaw et al, 2005;Katoh et al, 2006) and because somatic mutations have been identified in a subset of human lung tumors and cell lines (Sanchez-Cespedes et al, 2002;Ji et al, 2007), our data now suggest that enhanced Crtc gene family activity may participate in the tumorigenic process within LKB1 null tumors that are not otherwise associated with the recurrent t(11;19) rearrangement. Interestingly, overexpression of NR4A2 had been previously reported for HeLa and H460 cell lines, which are both known to be LKB1 null (Tiainen et al, 1999;Katoh et al, 2006). NR4A2 has also been tested as a candidate tumor suppressor gene where siRNA knock-down in HeLa cells was associated with parameters of growth inhibition and apoptosis (Ke et al, 2004).…”

“…We and others had previously shown that Crtc1 is a bona fide cancer gene when activated by a t(11;19) chromosomal translocation resulting in the aberrant transcription of certain Crtc1 inducible genes, including the orphan nuclear receptor NR4A2 (Tonon et al, 2003;Coxon et al, 2005;Wu et al, 2005). As LKB1 has been identified as a key regulator for Crtc2 transcriptional activity (Shaw et al, 2005;Katoh et al, 2006) and because somatic mutations have been identified in a subset of human lung tumors and cell lines (Sanchez-Cespedes et al, 2002;Ji et al, 2007), our data now suggest that enhanced Crtc gene family activity may participate in the tumorigenic process within LKB1 null tumors that are not otherwise associated with the recurrent t(11;19) rearrangement. Interestingly, overexpression of NR4A2 had been previously reported for HeLa and H460 cell lines, which are both known to be LKB1 null (Tiainen et al, 1999;Katoh et al, 2006).…”

“…Although AMPK, Sik2 and Mark2 have been proposed as major proximate regulators for Crtc2 (Lizcano et al, 2004;Dentin et al, 2007;Fu and Screaton, 2008;Hezel and Bardeesy, 2008), Sik1 has also been suggested as an important kinase inhibitor for Crtc1 transcriptional activity (Katoh et al, 2006). Further, SIK1 is a Crtc1/ cAMP/CREB target gene whose induction is proposed to phosphorylate and mediate a negative feedback loop on Crtc1 in neuronal cells .…”

Identification of salt-inducible kinase 3 as a novel tumor antigen associated with tumorigenesis of ovarian cancer

“…LKB1, has been well-documented to be a master upstream activator of AMPK and AMPK-related kinase, activates SIK1, SIK2 and SIK3 by phosphorylation of Thr182, Thr175 and Thr163 residues at their activation loops, respectively (Lizcano et al, 2004). Moreover, the work of Katoh et al (2006) showed the cytoplasmic retention of TORC2 could only be found in the LKB1-deficient HeLa cells when LKB1 was co-transfected with SIK3 into the cell. On the other hand, one point mutation of threonine to glutamate at the 163th residue in SIK3 (T163E) led to constitutive activation of SIK3, which was showed to be sufficient to cause the nuclear export of TORC2 and inactivate cAMP-response element …”

Altered LKB1/CREB-regulated transcription co-activator (CRTC) signaling axis promotes esophageal cancer cell migration and invasion

“…CRTC1 function thus becomes aberrantly activated, which contributes to CRTC1-MAML2 oncogenic activities (Coxon et al, 2005;Wu et al, 2005). CRTC1 co-activator function is attenuated by salt-induced kinases (SIKs), because SIKs phosphorylate CRTC1, preventing CRTC1 from entering the nucleus and activating CREB transcription (Katoh et al, 2006). As SIKs enzymatic activities are dependent on LKB1, it is anticipated that LKB1 deregulation impacts CRTC1 functional activities.…”