Protein kinase CK2 (also known as casein kinase 2) is present in the cytoplasm, nuclei, and several other organelles. In addition, this enzyme has been found bound to the external side of the cell membrane where it acts as an ectokinase phosphorylating several extracellular proteins. Previous experiments with transfection of HEK-293T cells demonstrated that expression of both subunits, CK2␣ (catalytic) and CK2␤ (regulatory), was necessary for the appearance of the ectopic enzyme as an ectokinase. In this work, using deletion and point mutations of CK2␤, it was possible to demonstrate that the region between amino acids 20 and 33 was necessary for the export of the enzyme as an ectokinase. Phenylalanines 21 and 22 and acidic residues in positions 26 -28 are involved in the structural aspects that are required for export. However, the region encompassing amino acids 20 -33 of CK2␤ is not sufficient to make the carboxyl half of this subunit functional in bringing CK2 to the ectokinase locus. In cells transfected with only CK2␤, it was demonstrated that 3-4% of the subunit is exported to the cell medium, but the subunit is not bound to the external membrane.casein kinase 2 ͉ export of proteins ͉ shedding I t has been known for a number of years that protein kinase CK2 (formerly called casein kinase 2) is present in the cytoplasm, nuclei, and several other cell organelles. It has also been found on the external side of the cellular membrane where, acting as an ectokinase, it can phosphorylate extracellular proteins and external domains of proteins. Kubler et al. (1) originally discovered that incubation of cells with CK2 substrates such as casein or phosvitin resulted in the liberation of the enzyme activity into the medium. This substrate-mediated release from the cell membrane was called ''shedding.'' Several extracellular proteins have been identified as substrates of the CK2 ectokinase activity. Vitronectin has been shown to be phosphorylated extracellularly by CK2, and evidence has been presented to demonstrate that its phosphorylation regulates the adhesion of cells to the extracellular matrix (2, 3). More recently, it has been shown that CK2 phosphorylates the C9 complement and it has been suggested that this phosphorylation might control cell lysis caused by this complement protein (4). Another recent example of an extracellular domain phosphorylated by ecto CK2 is the collagen XVII receptor. This phosphorylation may inhibit its degradation catalyzed by metaloproteases (5).In our previous work (6), we explored the conditions and requirements necessary to observe the presence of ectopically expressed CK2 holoenzyme that could be obtained after shedding of human cells in culture that had been transfected with the cDNAs coding for the two CK2 subunits. This work demonstrated that transfection with both catalytic (CK2␣) and regulatory (CK2␤) subunits was necessary to detect ectopically expressed CK2 bound externally to the cellular membrane. It was further shown that the export outside the cell did not require c...

The main component of the amyloid senile plaques found in Alzheimer's brain is the amyloid-beta-peptide (A beta), a proteolytic product of a membrane precursor protein. Previous structural studies have found different conformations for the A beta peptide depending on the solvent and pH used. In general, they have suggested an alpha-helix conformation at the N-terminal domain and a beta-sheet conformation for the C-terminal domain. The structure of the complete A beta peptide (residues 1-40) solved by NMR has revealed that only helical structure is present in A beta. However, this result cannot explain the large beta-sheet A beta aggregates known to form amyloid under physiological conditions. Therefore, we investigated the structure of A beta by molecular modeling based on extensive homology using the Smith and Waterman algorithm implemented in the MPsrch program (Blitz server). The results showed a mean value of 23% identity with selected sequences. Since these values do not allow a clear homology to be established with a reference structure in order to perform molecular modeling studies, we searched for detailed homology. A 28% identity with an alpha/beta segment of a triosephosphate isomerase (TIM) from Culex tarralis with an unsolved three-dimensional structure was obtained. Then, multiple sequence alignment was performed considering A beta, TIM from C.tarralis and another five TIM sequences with known three-dimensional structures. We found a TIM segment with secondary structure elements in agreement with previous experimental data for A beta. Moreover, when a synthetic peptide from this TIM segment was studied in vitro, it was able to aggregate and to form amyloid fibrils, as established by Congo red binding and electron microscopy. The A beta model obtained was optimized by molecular dynamics considering ionizable side chains in order to simulate A beta in a neutral pH environment. We report here the structural implications of this study.