Isoform specific phosphorylation of p53 by protein kinase CK1.

The ability of three isoforms of protein kinase CK1 (alpha, gamma(1), and delta) to phosphorylate the N-terminal region of p53 has been assessed using either recombinant p53 or a synthetic peptide reproducing its 1-28 sequence. Both substrates are readily phosphoylated by CK1delta and CK1alpha, but not by the gamma isoform. Affinity of full size p53 for CK1 is 3 orders of magnitude higher than that of its N-terminal peptide (K (m) 0.82 muM vs 1.51 mM). The preferred target is S20, whose phosphorylation critically relies on E17, while S6 is unaffected despite displaying the same consensus (E-x-x-S). Our data support the concept that non-primed phosphorylation of p53 by CK1 is an isoform-specific reaction preferentially affecting S20 by a mechanism which is grounded both on a local consensus and on a remote docking site mapped to the K(221)RQK(224) loop according to modeling and mutational analysis.

Chemical dissection of the APC Repeat 3 multistep phosphorylation by the concerted action of protein kinases CK1 and GSK3.

A crucial event in machinery controlled by Wnt signaling is the association of beta-catenin with the adenomatous polyposis coli (APC) protein, which is essential for the degradation of beta-catenin and requires the multiple phosphorylation of APC at six serines (1501, 1503, 1504, 1505, 1507, and 1510) within its repeat three (R3) region. Such a phosphorylation is believed to occur by the concerted action of two protein kinases, CK1 and GSK3, but its mechanistic aspects are a matter of conjecture. Here, by combining the usage of variably phosphorylated peptides reproducing the APC R3 region and Edman degradation assisted localization of residues phosphorylated by individual kinases, we show that the process is initiated by CK1, able to phosphorylate S1510 and S1505, both specified by non-canonical determinants. Phosphorylation of S1505 primes subsequent phosphorylation of S1501 by GSK3. In turn, phospho-S1501 triggers the hierarchical phosphorylation of S1504 and S1507 by CK1. Once phosphorylated, S1507 primes the phosphorylation of both S1510 and S1503 by CK1 and GSK3, respectively, thus completing all six phosphorylation steps. Our data also rule out the intervention of CK2 despite the presence of a potential CK2 phosphoacceptor site, S1510LDE, in the R3 repeat. S1510 is entirely unaffected by CK2, while it is readily phosphorylated even in the unprimed peptide by CK1delta but not by CK1gamma. This discloses a novel motif significantly different from non-canonical sequences phosphorylated by CK1 in other proteins, which appears to be specifically recognized by the delta isoform of CK1.

Bcr-Abl stabilizes beta-catenin in chronic myeloid leukemia through its tyrosine phosphorylation.

Self-renewal of Bcr-Abl(+) chronic myeloid leukemia (CML) cells is sustained by a nuclear activated serine/threonine-(S/T) unphosphorylated beta-catenin. Although beta-catenin can be tyrosine (Y)-phosphorylated, the occurrence and biological relevance of this covalent modification in Bcr-Abl-associated leukemogenesis is unknown. Here we show that Bcr-Abl levels control the degree of beta-catenin protein stabilization by affecting its Y/S/T-phospho content in CML cells. Bcr-Abl physically interacts with beta-catenin, and its oncogenic tyrosine kinase activity is required to phosphorylate beta-catenin at Y86 and Y654 residues. This Y-phospho beta-catenin binds to the TCF4 transcription factor, thus representing a transcriptionally active pool. Imatinib, a Bcr-Abl antagonist, impairs the beta-catenin/TCF-related transcription causing a rapid cytosolic retention of Y-unphosphorylated beta-catenin, which presents an increased binding affinity for the Axin/GSK3beta complex. Although Bcr-Abl does not affect GSK3beta autophosphorylation, it prevents, through its effect on beta-catenin Y phosphorylation, Axin/GSK3beta binding to beta-catenin and its subsequent S/T phosphorylation. Silencing of beta-catenin by small interfering RNA inhibited proliferation and clonogenicity of Bcr-Abl(+) CML cells, in synergism with Imatinib. These findings indicate the Bcr-Abl triggered Y phosphorylation of beta-catenin as a new mechanism responsible for its protein stabilization and nuclear signalling activation in CML.

The first armadillo repeat is involved in the recognition and regulation of beta-catenin phosphorylation by protein kinase CK1.

Multiple phosphorylation of beta-catenin by glycogen synthase kinase 3 (GSK3) in the Wnt pathway is primed by CK1 through phosphorylation of Ser-45, which lacks a typical CK1 canonical sequence. Synthetic peptides encompassing amino acids 38-64 of beta-catenin are phosphorylated by CK1 on Ser-45 with low affinity (K(m) approximately 1 mM), whereas intact beta-catenin is phosphorylated at Ser-45 with very high affinity (K(m) approximately 200 nM). Peptides extended to include a putative CK1 docking motif (FXXXF) at 70-74 positions or a F74AA mutation in full-length beta-catenin had no significant effect on CK1 phosphorylation efficiency. beta-Catenin C-terminal deletion mutants up to residue 181 maintained their high affinity, whereas removal of the 131-181 fragment, corresponding to the first armadillo repeat, was deleterious, resulting in a 50-fold increase in K(m) value. Implication of the first armadillo repeat in beta-catenin targeting by CK1 is supported in that the Y142E mutation, which mimics phosphorylation of Tyr-142 by tyrosine kinases and promotes dissociation of beta-catenin from alpha-catenin, further improves CK1 phosphorylation efficiency, lowering the K(m) value to <50 nM, approximating the physiological concentration of beta-catenin. In contrast, alpha-catenin, which interacts with the N-terminal region of beta-catenin, prevents Ser-45 phosphorylation of CK1 in a dose-dependent manner. Our data show that the integrity of the N-terminal region and the first armadillo repeat are necessary and sufficient for high-affinity phosphorylation by CK1 of Ser-45. They also suggest that beta-catenin association with alpha-catenin and beta-catenin phosphorylation by CK1 at Ser-45 are mutually exclusive.

Generation of protein kinase Ck1alpha mutants which discriminate between canonical and non-canonical substrates.

Protein kinase CK1 denotes a family of pleiotropic serine/threonine protein kinases implicated in a variety of cellular functions. Typically, CK1 acts as a 'phosphate-directed' kinase whose targeting is primed by a single phosphorylated side chain at position n-3 or n-4 relative to serine/threonine, but increasing evidence is accumulating that CK1 can also engage some of its substrates at sites that do not conform to this canonical consensus. In the present paper, we show that CK1a phosphorylates with the same efficiency phosphopeptides primed by a phosphoserine residue at either n-3 [pS(-3)] or n-4 [pS(-4)] positions. The phosphorylation efficiency of the pS(-4) peptide, and to a lesser extent that of the pS(-3) peptide, is impaired by the triple mutation of the lysine residues in the K229KQK232 stretch to alanine residues, promoting 40-fold and 6-fold increases of Km respectively. In both cases, the individual mutation of Lys232 is as detrimental as the triple mutation. A kinetic alanine-scan analysis with a series of substituted peptide substrates in which the priming phosphoserine residue was effectively replaced by a cluster of four aspartate residues was also consistent with a crucial role of Lys232 in the recognition of the acidic determinant at position n-4. In sharp contrast, the phosphorylation of b-catenin and of a peptide including the non-canonical b-catenin site (Ser45) lacking acidic/phosphorylated determinants upstream is not significantly affected by mutations in the KKQK stretch. These data provide a molecular insight into the structural features that underlie the site specificity of CK1a and disclose the possibility of developing strategies for the preferential targeting of subsets of CK1 substrates.

Basic region of residues 228-231 of protein kinase CK1alpha is involved in its interaction with axin: binding to axin does not affect the kinase activity.

Protein kinase CK1, also known as casein kinase 1, participates in the phosphorylation of beta-catenin, which regulates the functioning of the Wnt signaling cascade involved in embryogenesis and carcinogenesis. beta-catenin phosphorylation occurs in a multiprotein complex assembled on the scaffold protein axin. The interaction of CK1alpha from Danio rerio with mouse-axin has been studied using a pull-down assay that uses fragments of axin fused to glutathione S transferase, which is bound to glutathione sepharose beads. The results indicate that the three lysines present in the basic region of residues 228-231 of CK1alpha are necessary for the binding of CK1 to axin. Lysine 231 is particularly important in this interaction. In order to define the relevance of the axin-CK1alpha interaction, the effect of the presence of axin on the phosphorylating activity of CK1alpha was tested. It is also evident that the region of axin downstream of residues 503-562 is required for CK1alpha interaction. The binding of CK1alpha to axin fragment 292-681 does not facilitate the phosphorylation of beta-catenin despite the fact that this axin fragment can also bind beta-catenin. Binding of CK1alpha to axin is not required for the phosphorylation of axin itself and, likewise, axin does not affect the kinetic parameters of the CK1alpha towards casein or a specific peptide substrate.

A noncanonical sequence phosphorylated by casein kinase 1 in beta-catenin may play a role in casein kinase 1 targeting of important signaling proteins.

Protein kinase casein kinase 1 (CK1) phosphorylates Ser-45 of beta-catenin, "priming" the subsequent phosphorylation by glycogen synthase-3 of residues 41, 37, and 33. This concerted phosphorylation of beta-catenin signals its degradation and prevents its function in triggering cell division. The sequence around Ser-45 does not conform to the canonical consensus for CK1 substrates, which prescribes either phosphoamino acids or acidic residues in position n-3 from the target serine. However, the beta-catenin sequence downstream from Ser-45 is very similar to a sequence recognized by CK1 in nuclear factor for activated T cells 4. The common features include an SLS motif followed two to five residues downstream by a cluster of acidic residues. Synthetic peptides reproducing residues 38-65 of beta-catenin were assayed with purified rat liver CK1 or recombinant CK1 alpha and CK1 alpha L from zebrafish. The results demonstrate that SLS and acidic cluster motifs are crucial for CK1 recognition. Pro-44 and Pro-52 are also important for efficient phosphorylation. Similar results were obtained with the different isoforms of CK1. Phosphorylation of mutants of full-length recombinant beta-catenin from zebrafish confirmed the importance of the SLS and acidic cluster motifs. A search for proteins with similar motifs yielded, among other proteins, adenomatous polyposis coli, previously found to be phosphorylated by CK1. There is a strong correlation of beta-catenin mutations found in thyroid tumors with the motifs recognized by CK1 in this protein.