Four-gene expression model predictive of lymph node metastases in oral squamous cell carcinoma.

BACKGROUND: Previous knowledge of cervical lymph node compromise may be crucial to choose the best treatment strategy in oral squamous cell carcinoma (OSCC). Here we propose a set four genes, whose mRNA expression in the primary tumor predicts nodal status in OSCC, excluding tongue. MATERIAL AND METHODS: We identified differentially expressed genes in OSCC with and without compromised lymph nodes using Differential Display RT-PCR. Known genes were chosen to be validated by means of Northern blotting or real time RT-PCR (qRT-PCR). Thereafter we constructed a Nodal Index (NI) using discriminant analysis in a learning set of 35 patients, which was further validated in a second independent group of 20 patients. RESULTS: Of the 63 differentially expressed known genes identified comparing three lymph node positive (pN +) and three negative (pN0) primary tumors, 23 were analyzed by Northern analysis or RT-PCR in 49 primary tumors. Six genes confirmed as differentially expressed were used to construct a NI, as the best set predictive of lymph nodal status, with the final result including four genes. The NI was able to correctly classify 32 of 35 patients comprising the learning group (88.6%; p = 0.009). Casein kinase 1alpha1 and scavenger receptor class B, member 2 were found to be up regulated in pN + group in contrast to small proline-rich protein 2B and Ras-GTPase activating protein SH3 domain-binding protein 2 which were upregulated in the pN0 group. We validated further our NI in an independent set of 20 primary tumors, 11 of them pN0 and nine pN + with an accuracy of 80.0% (p = 0.012). CONCLUSIONS: The NI was an independent predictor of compromised lymph nodes, taking into the consideration tumor size and histological grade. The genes identified here that integrate our "Nodal Index" model are predictive of lymph node metastasis in OSCC.

Autophosphorylation of carboxy-terminal residues inhibits the activity of protein kinase CK1alpha.

CK1 constitutes a protein kinase subfamily that is involved in many important physiological processes. However, there is limited knowledge about mechanisms that regulate their activity. Isoforms CK1delta and CK1epsilon were previously shown to autophosphorylate carboxy-terminal sites, a process which effectively inhibits their catalytic activity. Mass spectrometry of CK1alpha and splice variant CK1alphaL has identified the autophosphorylation of the last four carboxyl-end serines and threonines and also for CK1alphaS, the same four residues plus threonine-327 and serine-332 of the S insert. Autophosphorylation occurs while the recombinant proteins are expressed in Escherichia coli. Mutation of four carboxy-terminal phosphorylation sites of CK1alpha to alanine demonstrates that these residues are the principal but not unique sites of autophosphorylation. Treatment of autophosphorylated CK1alpha and CK1alphaS with lambda phosphatase causes an activation of 80-100% and 300%, respectively. Similar treatment fails to stimulate the CK1alpha mutants lacking autophosphorylation sites. Incubation of dephosphorylated enzymes with ATP to allow renewed autophosphorylation causes significant inhibition of CK1alpha and CK1alphaS. The substrate for these studies was a synthetic canonical peptide for CK1 (RRKDLHDDEEDEAMS*ITA). The stimulation of activity seen upon dephosphorylation of CK1alpha and CK1alphaS was also observed using the known CK1 protein substrates DARPP-32, beta-catenin, and CK2beta, which have different CK1 recognition sequences. Autophosphorylation effects on CK1alpha activity are not due to changes in Km(app) for ATP or for peptide substrate but rather to the catalytic efficiency per pmol of enzyme. This work demonstrates that CK1alpha and its splice variants can be regulated by their autophosphorylation status.

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.