br Transparency document br Results and Discussion br Experi
Results and Discussion
The casein kinase 1 (CK1) family consists of at least seven different gene products, often referred to as isoforms (α, β, γ1, γ2, γ3, δ, ε, with splice forms described for CK1α, γ, and ε (reviewed in ). This diversification is in agreement with the variety of cellular processes in which this kinase family is involved. CK1α is a priming kinase in the phosphorylation-dependent degradation of β-catenin, an important process during Wnt signalling . This signalling pathway is activated in different types of cancer, indicating a role of CK1 in cancer development. CK1δ and ε are implicated in p53-dependent apoptosis and mitotic checkpoint control , providing another link of CK1 to cancer. CK1α, δ, and ε are overexpressed in brain regions of Alzheimer’s disease patients and tightly associate with granulovacuolar degeneration bodies as well as with neurofibrillary tangles and amyloid plaques . The circadian clock that influences a variety of body functions and also the course of diseases (reviewed in ) is partly regulated by CK1δ and ε by phosphorylating Per proteins . Apart from the CK1 isoforms γ3, δ, and ε, which are negatively regulated by autophosphorylation , , , CK1 isoforms are thought to be constitutively active and their regulation is thought to be at least partly mediated by localisation and/or complex formation (reviewed in ), which can be controlled by scaffolding proteins. One of these scaffolding proteins is axin, the product of a gene originally named “fused” , which interacts with the CK1 isoforms α, δ, and ε (reviewed in ). Axin is a scaffolding protein that is implicated in the Wnt signalling pathway as well as in JNK activation (reviewed in ). Among the axin-binding proteins so far identified are the adenomatous polyposis coli protein (APC), β-catenin, CK1, glycogen synthase kinase 3 beta (GSK-3β), and the protein phosphatase PP2A, which are involved in Wnt signalling. Moreover, axin can dimerise via the Dix domain, which also allows binding of the dishevelled protein (reviewed in ) (see a). The axin residues responsible for the binding to GSK-3β were identified to be BLZ945 353–437 . This information was used by our group to construct an axin fragment from amino acid 290 to 544 to purify recombinant rat GSK-3β as well as native GSK-3 from yeast, sea urchin embryos, porcine brain and mammalian cultured cells . Two regions of axin have been described to be responsible for CK1 binding: the region between amino acids 217 and 352 and the region carboxy terminal to amino acid 507 . The data indicated that the carboxy-terminal binding domain was sufficient for CK1α binding, whereas the presence of both binding domains was necessary for CK1ε binding. The amino-terminal region was not sufficient to bind either CK1α or CK1ε . The carboxy-terminal binding site for CK1α was further characterised to reside between the amino acids 503 and 684, and to interact with the basic amino acids 228–231 of CK1α (KKQK) from the zebrafish . It was shown elsewhere that a carboxy terminal fragment of axin comprising the amino acids 475–676, is also able to bind to CK1ε, albeit with less efficiency than a fragment from amino acids 284 to 712, comprising both CK1 binding domains . The basic amino acid stretch KKQK from human CK1α (229–232) is also present in CK1ε and CK1δ (data not shown), confirmatory to their ability to bind to axin. Interestingly, a blastp search using the human CK1α sequence also found this sequence in the predicted CK1α and CK1δ sequence of the sea urchin , as well as in the CK1 homolog HRR25p of (data not shown).
Introduction The phosphorylation–dephosphorylation cycle controls the activity of a large number of proteins, including several members of the Ras superfamily of low molecular weight GTP-binding proteins. Small GTPases act as molecular switches orchestrating a variety of signalling pathways by cycling between an inactive GDP-bound form to an GTP-bound active form . Emerging reports suggest that phosphorylation of small GTPases contributes to their activity regulation, most often independently of GTP/GDP cycling and was proposed as a secondary switch in addition to GDP/GTP cycle. For instance, while the phosphorylation of Rac1 or Rab6 modifies their affinity for GTP , , the phosphorylation of K-Ras by PKA or PKC leads to its activation but without impact on the GTP binding or GTPase activity . Phosphorylation interferes with functions of several small G proteins by inducing their translocation from membrane to cytosolic compartments as in the case of Rap1A , RhoE  Cdc42 or RhoA , , .