CASK-interactive protein1 is a newly recognized post-synaptic density protein in mammalian neurons. Although its N-terminal region contains several well-known functional domains, its entire C-terminal proline-rich region of 800 amino acids lacks detectable sequence homology to any previously characterized protein. We used multiple techniques for the structural characterization of this region and its three fragments. By bioinformatics predictions, CD spectroscopy, wide-line and 1 H-NMR spectroscopy, limited proteolysis and gel filtration chromatography, we provided evidence that the entire proline-rich region of CASK-interactive protein1 is intrinsically disordered. We also showed that the proline-rich region is biochemically functional, as it interacts with the adaptor protein Abl-interactor-2. To extend the finding of a high level of disorder in this scaffold protein, we collected 74 scaffold proteins (also including proteins denoted as anchor and docking), and predicted their disorder by three different algorithms. We found that a very high fraction (53.6% on average) of the residues fall into local disorder and their ordered domains are connected by linker regions which are mostly disordered (64.5% on average). Because of this high frequency of disorder, the usual design of scaffold proteins of short globular domains (86 amino acids on average) connected by longer linker regions (140 amino acids on average) and the noted binding functions of these regions in both CASK-interactive protein1 and the other proteins studied, we suggest that structurally disordered regions prevail and play key recognition roles in scaffold proteins.

Systemic and local cytokine environment may modulate the immunogenicity of colorectal cancer cells, and affect anti-tumor immune functions of tumor-infiltrating lymphocytes. We therefore investigated cytokine mRNA expression patterns in tumors and peripheral blood mononuclear cells (PBMC) from patients with colorectal adenocarcinoma. IL-2, IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), IL-4, IL-6, IL-8, IL-10 and IL-1 beta mRNAs in single cell suspension of freshly isolated colorectal cancer tissue were studied by RT-PCR. Frequencies of cytokine gene expression were compared to those in normal colonic mucosa from tumor patients. The frequencies of IL-2, IFN-gamma, IL-4 and IL-10 gene expression were also determined in peripheral blood mononuclear cells from patients with colorectal adenocarcinoma and compared to those of healthy individuals. Tumor samples were more frequently positive for IFN-gamma, IL-2, TNF-alpha and IL-10 gene expression than normal mucosa (p=0.0001, p=0.0118, p=0.001 and p<0.0001, respectively). Frequencies of IL-2 and TNF-alpha gene expressions were significantly higher in tumors with a diameter <5 cm, than in those with a diameter >5 cm. The genes for IL-6, IL-1 beta and IL-8 were commonly expressed in both tumor tissue and normal colonic mucosa. IFN-gamma transcripts were detected in more PBMC samples from patients with colorectal cancer than those from normal controls (p=0.0449). Thus, colorectal cancer tissue is characterized by a specific pattern of cytokine gene expression. It is likely that multiple interactions between pro- and anti-inflammatory cytokines regulate tumor growth and the functional activity of tumor-infiltrating lymphocytes.

BackgroundScaffold proteins have an important role in the regulation of signal propagation. These proteins do not possess any enzymatic activity but can contribute to the formation of multiprotein complexes. Although scaffold proteins are present in all cell types, the nervous system contains them in the largest amount. Caskin proteins are typically present in neuronal cells, particularly, in the synapses. However, the signaling mechanisms by which Caskin proteins are regulated are largely unknown.ResultsHere we demonstrate that EphB1 receptor tyrosine kinase can recruit Caskin1 through the adaptor protein Nck. Upon activation of the receptor kinase, the SH2 domain of Nck binds to one of its tyrosine residues, while Nck SH3 domains interact with the proline-rich domain of Caskin1. Complex formation of the receptor, adaptor and scaffold proteins results in the tyrosine phosphorylation of Caskin1 on its SH3 domain. The phosphorylation sites were identified by mass-spectrometry as tyrosines 296 and 336. To reveal the structural consequence of this phosphorylation, CD spectroscopy was performed. This measurement suggests that upon tyrosine phosphorylation the structure of the Caskin1 SH3 domain changes significantly.ConclusionTaken together, we propose that the scaffold protein Caskin1 can form a complex with the EphB1 tyrosine kinase via the Nck protein as a linker. Complex formation results in tyrosine phosphorylation of the Caskin1 SH3 domain. Although we were not able to identify any physiological partner of the SH3 domain so far, we could demonstrate that phosphorylation on conserved tyrosine residues results in marked changes in the structure of the SH3 domain.

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