Deletions of portions of chromosomes 1p and 19q are closely associated with the oligodendroglioma histologic phenotype. In most cases, 1p and 19q are codeleted, yet the mechanism of dual loss is unexplained. We report 5 cases (World Health Organization grade III) in which metaphase cytogenetics identified a derivative chromosome consisting of what appears to be the whole arms of 1q and 19p forming a der(1;19)(q10;p10). Metaphase fluorescent in situ hybridization (FISH) confirmed the derivative chromosome was composed of 1q and 19p material in 3 cases; in 2 cases with few metaphases, FISH confirmed 19p material on the derivative chromosome. In all cases, interphase FISH showed net loss of 1p and 19q in 77% to 92% of cells, and microsatellite studies were consistent with 1p and 19q loss. We hypothesize the following: occurrence of a balanced whole-arm translocation between chromosomes 1 and 19 forming 2 derivative chromosomes, one composed of 1q and 19p, the other of 1p and 19q. Subsequent loss of the der(1;19)(p10;q10) then results in the simultaneous 1p and 19q loss observed in oligodendroglioma with retention of the der(1;19)(q10;p10) seen in these cases.
The myristoylated alanine-rich C kinase substrate (MARCKS) protein, a prominent cellular substrate for protein kinase C, is associated with membranes in various cell types. MARCKS is myristoylated at its amino terminus; this modification is thought to play the major role in anchoring MARCKS to cellular membranes. Recent studies have suggested that the protein's basic phosphorylation site/calmodulin binding domain may also be involved in the membrane association of MARCKS through electrostatic interactions. The present studies used mutations in the primary structure of the protein to investigate the nature of the association between MARCKS and cell membranes. In chick embryo fibroblasts, activation of protein kinase C led to a decrease in MARCKS membrane association as determined by cell fractionation techniques. Cell-free assays revealed that nonmyristoylated MARCKS exhibited almost no affinity for fibroblast membranes, despite readily demonstrable binding of the wild-type protein. Similar experiments in which the four serines in the phosphorylation site domain were mutated to aspartic acids, mimicking phosphorylation, decreased, but did not eliminate, membrane binding when compared to either the wild-type protein or a comparable tetra-asparagine mutant. Addition of calmodulin in the presence of Ca2+ also inhibited binding of the wild-type protein to membranes, presumably by neutralizing the phosphorylation site domain, or by physically interfering with its membrane association. Surprisingly, expression of a nonmyristoylatable mutant form of MARCKS in intact cells led to only a 46% decrease in its plasma membrane association, as determined by cell fractionation and immunoelectron microscopy. These results are consistent with a complex model of the interaction of MARCKS with cellular membranes, in which the myristoyl moiety, the positively charged phosphorylation site domain, and possibly other domains make independent contributions to membrane binding in intact cells.
The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a widely expressed, prominent substrate for protein kinase C. MARCKS is largely associated with membranes in cells, and hydrophobic interactions involving the amino-terminal myristoyl moiety are thought to play a role in anchoring MARCKS to cellular membranes. In addition, experiments in cell-free systems have suggested that electrostatic interactions between the positively charged phosphorylation site/calmodulin binding domain (PSD) of MARCKS and negatively charged membrane lipids are also involved in this association. Although it has been inferred from phosphorylation experiments, the electrostatic nature of the interaction between the PSD and membranes has not been demonstrated directly in intact cells. We expressed human MARCKS mutated in the myristoylation site and the PSD in REF52 cells; the cells were then fractionated by ultracentrifugation. Both nonmyristoylatable MARCKS and MARCKS in which the four serines in the PSD were mutated to aspartic acids, mimicking phosphorylation, exhibited decreased membrane affinity when compared to the fully myristoylated, wild-type, tetra-Ser protein or a myristoylated, tetra-Asn mutant. A double mutant, nonmyristoylatable protein in which the four serines in the PSD were mutated to aspartic acids exhibited negligible membrane association. Similar results were obtained in 293 cells that stably expressed chicken MARCKS mutated in the same domains. The double mutant, nonmyristoylatable tetra-Asp chicken protein exhibited little membrane association as determined by both subcellular fractionation and immunoelectron microscopy. These results indicate that myristoylation and electrostatic interactions involving the PSD exert independent, essentially additive effects on the membrane association of MARCKS in intact cells.
The myristoylated alanine-rich C kinase substrate, or MARCKS protein, is a widely expressed, prominent substrate for protein kinase C. Although the exact function of MARCKS has not been elucidated, targeted disruption of the MARCKS gene (Macs) in mice has shown that MARCKS plays a crucial role in the development of the central nervous system. Mice deficient in MARCKS exhibited universal perinatal death with defects in neurulation, fusion of the cerebral hemispheres, formation of the great forebrain commissures, and retinal and cortical lamination (Stumpo et al., Proc. Natl. Acad. Sci. USA 92, 944-948, 1995). In the present studies, a transgene consisting of approximately 3.4 kb of promoter from the human MARCKS gene (MACS), with an epitope tag sequence inserted at the carboxyl terminus of the MARCKS coding region, was able to complement completely MARCKS deficiency in mice. Thus, the human transgene contained all of the elements necessary for normal developmental expression of MARCKS. To test the importance of MARCKS myristoylation to its developmental role, an otherwise identical transgene was constructed in which the glycine at the amino terminus of MARCKS was mutated to an alanine. This mutation, which resulted in the expression of nonmyristoylated MARCKS, was successful in partially rescuing the Macs null phenotype. Specifically, about 25% of these mice survived the perinatal period; these survivors appeared to develop normally except for slightly decreased body size. In both the survivors and the nonsurvivors, all of the known anatomical defects associated with MARCKS deficiency were corrected by expression of the nonmyristoylated human protein. These results indicate that myristoylation of MARCKS is not required for the protein to correct many of the developmental abnormalities characteristic of its deficiency.
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