Abstract
The transforming growth factor-beta (TGF-beta) signaling pathway is known to be involved in a wide range of biological events, including development, cellular differentiation, apoptosis, and oncogenesis. The TGF-beta signal is mediated by ligand binding to the type II receptor, leading to the recruitment and activation of the type I receptor, and subsequent activation of a family of intracellular signal transducing proteins called Smads. Here we report a regulatory role for casein kinase Iepsilon (CKIepsilon) in the TGF-beta signaling cascade. We find that CKIepsilon binds to all Smads and the cytoplasmic domains of the type I and type II receptors both in vitro and in vivo. The interaction of CKIepsilon with the type I and type II receptors is independent of TGF-beta stimulation, whereas the CKIepsilon/Smad interaction is transiently disrupted by ligand treatment. Additionally, CKIepsilon is able to phosphorylate the receptor-activated Smads (Smads 1-3 and 5) and the type II receptor in vitro. Transcriptional reporter assays reveal that transient overexpression of wild type CKIepsilon dramatically reduces basal reporter activity but enhances TGF-beta-stimulated transcription. Furthermore, overexpression of a kinase-dead mutant of CKIepsilon inhibits both basal and ligand-induced transcription, whereas inhibition of endogenous CKI catalytic activity with IC261 blocks only TGF-beta-stimulated reporter activity. Finally, knocking down CKIepsilon protein levels results in a significant increase in basal and TGF-beta-induced transcription. These results suggest that CKIepsilon plays a ligand-dependent, differential, and dual regulatory role within the TGF-beta signaling pathway.
Highlights
Homeostasis of organisms as diverse as fruit flies and humans
Knocking down casein kinase I (CKI)⑀ protein levels results in a significant increase in basal and TGF--induced transcription. These results suggest that CKI⑀ plays a liganddependent, differential, and dual regulatory role within the TGF- signaling pathway
By using Smad3 deletion mutants fused to GST and depicted in Fig. 1A, we found that CKI⑀ interacts most strongly with the MH2 domain
Summary
Homeostasis of organisms as diverse as fruit flies and humans. This pathway begins its regulatory functions at the earliest stages of development and acts to coordinate the complex mechanisms of cellular differentiation that will result in a mature organism. The casein kinase I (CKI) family consists of seven known isoforms (␣, , ␥1, ␥2, ␥3, ␦, and ⑀) that possess a highly homologous serine/threonine kinase domain and a divergent and variable length carboxyl-terminal tail [8, 9]. These kinases have been implicated in a wide range of cellular functions, including vesicular trafficking, DNA damage repair, cell cycle progression, and cytokinesis [8]. This idea is clearly supported by the numerous papers that have been published in the last several years that show a significant role for CKI⑀ and CKI␦ in the circadian rhythms of mammals [13,14,15,16,17,18,19], the regulation of a Gq/11-coupled receptor, the inhibition of the nuclear translocation of NF-AT4, the negative regulation of the Wnt and Hedgehog signaling pathways by CKI␣ (20 –28), the regulation of the -platelet-derived growth factor receptor by CKI␥2 [29], and the regulation of the Wnt and JNK signaling pathways by CKI⑀ (30 – 41)
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