Six infants in an Old Order Amish pedigree were observed to be affected with endocrine-cerebro-osteodysplasia (ECO). ECO is a previously unidentified neonatal lethal recessive disorder with multiple anomalies involving the endocrine, cerebral, and skeletal systems. Autozygosity mapping and sequencing identified a previously unknown missense mutation, R272Q, in ICK, encoding intestinal cell kinase (ICK). Our results established that R272 is conserved across species and among ethnicities, and three-dimensional analysis of the protein structure suggests protein instability due to the R272Q mutation. We also demonstrate that the R272Q mutant fails to localize at the nucleus and has diminished kinase activity. These findings suggest that ICK plays a key role in the development of multiple organ systems.

The convergence of caspase and protein kinase signaling pathways has become increasingly evident, as illustrated by the protection of caspase substrates from cleavage upon undergoing phosphorylation at or near to their caspase recognition motifs. To investigate the global role of phosphorylation in the regulation of caspase signaling, we designed a peptide match program to identify sequences from the human proteome that contained overlapping recognition motifs for caspases and kinases. We identified the protein kinase CK2 as the most prominent kinase with a consensus site for phosphorylation that overlapped with caspase recognition motifs. We then evaluated potential targets of CK2 and caspases by combining peptide array target screens with identification of caspase substrates. We identified numerous shared candidate targets of CK2 and caspases, including procaspase-3, which functions at a level at which both intrinsic and extrinsic apoptotic signals converge. Together, these data support a role for CK2-dependent phosphorylation as a global mechanism for inhibiting caspase signaling pathways.

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Phosphorylation is a crucial regulatory mechanism governing cellular signal transduction pathways, and despite the large number of identified sites to date, most mechanistic studies remain focused on individual phosphorylation sites. This study is the first to systematically determine specific consensus sequences for hierarchical phosphorylation events. The results indicate that individual phosphorylation sites should not be studied in isolation, and that larger, multisite phosphorylation motifs may have profound impact on cellular signaling. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.

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Protein kinases catalyze the phosphorylation of cellular proteins involved in the regulation of many cellular processes and have emerged as promising targets for the treatment of several diseases. Conventional assays to monitor protein kinase activity are limited because they typically rely on transfer of radioactive phosphate or phospho-specific antibodies that recognize specific substrates or sequence motifs. To overcome the limitations of conventional assays, we have developed a versatile approach based on transfer of ferrocene-phosphate that can be readily monitored using electrochemical detection or detection with antiferrocene antibodies in an immunoarray format. This assay is readily adapted to multiplex arrays and can be employed for monitoring kinase activity in complex mixtures and for kinase inhibitor profiling.

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Protein kinases have emerged as attractive targets for treatment of several diseases prompting large-scale phosphoproteomics studies to elucidate their cellular actions and the design of novel inhibitory compounds. Current limitations include extensive reliance on consensus predictions to derive kinase–substrate relationships from phosphoproteomics data and incomplete experimental validation of inhibitors. To overcome these limitations in the case of protein kinase CK2, we employed functional proteomics and chemical genetics to enable identification of physiological CK2 substrates and validation of CK2 inhibitors including TBB and derivatives. By 2D electrophoresis and mass spectrometry, we identified the translational elongation factor EEF1D as a protein exhibiting CK2 inhibitor-dependent decreases in phosphorylation in 32P-labeled HeLa cells. Direct phosphorylation of EEF1D by CK2 was shown by performing CK2 assays with EEF1D-FLAG from HeLa cells. Dramatic increases in EEF1D phosphorylation following λ–phosphatase treatment and phospho-EEF1D antibody recognizing EEF1D pS162 indicated phosphorylation at the CK2 site in cells. Furthermore, phosphorylation of EEF1D in the presence of TBB or TBBz is restored using CK2 inhibitor-resistant mutants. Collectively, our results demonstrate that EEF1D is a bona fide physiological CK2 substrate for CK2 phosphorylation. Furthermore, this validation strategy could be adaptable to other protein kinases and readily combined with other phosphoproteomic methods.