Real time fluorescence imaging of PLC gamma translocation and its interaction with the epidermal growth factor receptor.

The translocation of fluorescently tagged PLC gamma and requirements for this process in cells stimulated with EGF were analyzed using real time fluorescence microscopy applied for the first time to monitor growth factor receptor--effector interactions. The translocation of PLC gamma to the plasma membrane required the functional Src homology 2 domains and was not affected by mutations in the pleckstrin homology domain or inhibition of phosphatidylinositol (PI) 3-kinase. An array of domains specific for PLC gamma isoforms was sufficient for this translocation. The dynamics of translocation to the plasma membrane and redistribution of PLC gamma, relative to localization of the EGF receptor and PI 4,5-biphosphate (PI 4,5-P(2)), were shown. Colocalization with the receptor was observed in the plasma membrane and in membrane ruffles where PI 4,5-P(2) substrate could also be visualized. At later times, internalization of PLC gamma, which could lead to separation from the substrate, was observed. The data support a direct binding of PLC gamma to the receptor as the main site of the plasma membrane recruitment. The presence of PLC gamma in membrane structures and its access to the substrate appear to be transient and are followed by a rapid incorporation into intracellular vesicles, leading to downregulation of the PLC activity.

Catalytic domain of phosphoinositide-specific phospholipase C (PLC). Mutational analysis of residues within the active site and hydrophobic ridge of plcdelta1.

Structural studies of phospholipase C delta1 (PLCdelta1) in complexes with the inositol-lipid headgroup and calcium identified residues within the catalytic domain that could be involved in substrate recognition, calcium binding, and catalysis. In addition, the structure of the PLCdelta1 catalytic domain revealed a cluster of hydrophobic residues at the rim of the active site opening (hydrophobic ridge). To assess a role of each of these residues, we have expressed, purified, and characterized enzymes with the point mutations of putative active site residues (His311, Asn312, Glu341, Asp343, His356, Glu390, Lys438, Lys440, Ser522, Arg549, and Tyr551) and residues from the hydrophobic ridge (Leu320, Phe360, and Trp555). The replacements of most active site residues by alanine resulted in a great reduction (1,000-200,000-fold) of PLC activity analyzed in an inositol lipid/sodium cholate mixed micelle assay. Measurements of the enzyme activity toward phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4, 5-bis-phosphate (PIP2) identified Ser522, Lys438, and Arg549 as important for preferential hydrolysis of polyphosphoinositides, whereas replacement of Lys440 selectively affected only hydrolysis of PIP2. When PLC activity was analyzed at different calcium concentrations, substitutions of Asn312, Glu390, Glu341, and Asp343 resulted in a shift toward higher calcium concentrations required for PIP2 hydrolysis, suggesting that all these residues contribute toward Ca2+ binding. Mutational analysis also confirmed the importance of His311 ( approximately 20,000-fold reduction) and His356 ( approximately 6,000-fold reduction) for the catalysis. Mutations within the hydrophobic ridge, which had little effect on PIP2 hydrolysis in the mixed-micelles, resulted in an enzyme that was less dependent on the surface pressure when analyzed in a monolayer. This systematic mutational analysis provides further insights into the structural basis for the substrate specificity, requirement for Ca2+ ion, catalysis, and surface pressure/activity dependence, with general implications for eukaryotic phosphoinositide-specific PLCs.

Phospholipase C delta 1 requires a pleckstrin homology domain for interaction with the plasma membrane.

The structural requirements of phospholipase C delta 1 for interaction with the plasma membrane were analysed by immunofluorescence after microinjection into living cells. Microinjection of deletion mutants revealed that the region required for membrane attachment and binding of inositol 1,4,5-trisphosphate in vitro corresponded to the pleckstrin homology domain, a structural module described in more than 90 proteins.

Mutations within a highly conserved sequence present in the X region of phosphoinositide-specific phospholipase C-delta 1.

Phosphoinositide-specific phospholipase C (PI-PLC) enzymes have considerable structural similarity within limited regions (X and Y) implicated in catalysis. The role of residues contained within a highly conserved sequence present in the X region was investigated by site-directed mutagenesis of PLC-delta 1 isoenzyme. Seven residues (Ser-308, Ser-309, Ser-310, His-311, Thr-313, Tyr-314, and Gln-319) were individually replaced by alanine or glutamine (His-311). Replacement of two residues, His-311 and Tyr-314, resulted in a dramatic reduction of enzyme activity. The kcat of hydrolysis of phosphatidylinositol 4,5-bisphosphate by H311A and Y314A mutants was reduced 1000- and 10-fold respectively, with little effect on Km. Further analysis of H311A and Y314A mutants, using limited proteolysis and circular dichroism, had shown that no major structural alterations had occurred. Since site-directed mutagenesis demonstrated the importance of histidine residues, their role in enzyme function was also analysed by chemical modification with diethyl pyrocarbonate. This modification of histidine residues resulted in the reduction of enzyme activity and also indicated that more than one residue could be important.

Structural requirements of phosphatidylinositol-specific phospholipase C delta 1 for enzyme activity.

Phosphatidylinositol-specific phospholipase C delta 1 isozyme of phosphoinositol-specific phospholipase C has been used for studies of structural requirements for the catalytic function. The enzyme was expressed in a bacterial system and purified to homogeneity. Using a combination of deletion mutant analysis and limited proteolysis, it was found that the large proportion of the molecule participated in formation of a catalytic domain (residues 139-756); it included regions of high and low conservation with other phospholipase-C molecules. These studies also showed that the residues spanning regions of conservation, designated as X and Y, were exposed and highly susceptible to proteolysis by trypsin. Two of the fragments resulting from the cleavage (30 kDa and 40 kDa) interacted and, under non-denaturing conditions, formed a protein of 70 kDa.

Construct validity of the Commitment to Career Choices Scale among late adolescents: another look.

In response to Stead and Watson (1992), we reassessed the construct validity of the Commitment to Career Choices Scale using an age-constrained sample from our aggregate data base. A confirmatory factor analysis yielded findings comparable to Stead and Watson's results, thereby suggesting that their findings were likely due to using a homogeneous sample with truncated scores. Given the theoretical importance of measuring the full range of the two commitment to career choices continua, it seems premature to delete items from the scales for research with South African university students. Alternatively, we encourage the use of norms to interpret scores from the scales.

Activated Ki-ras genes in bladder epithelial cell lines transformed by treatment of primary mouse bladder explant cultures with 7,12-dimethylbenz[a]anthracene.

DNA from five lines of transformed bladder epithelial cells derived from cultures of primary cells that had been treated with 7,12-dimethylbenz[a]anthracene (DMBA) can transform NIH 3T3 mouse fibroblasts in DNA transfection experiments. Southern analysis of DNA from NIH 3T3 primary and secondary transformants established that four of the DMBA-transformed cell lines contained activated cellular Ki-ras, while the remaining cell line contained a transforming gene that is unrelated to Ki-ras, N-ras, and Ha-ras. The point mutations responsible for Ki-ras activation were detected using oligonucleotide probes following selective amplification of Ki-ras specific sequences using the polymerase chain reaction. The results showed that activation of Ki-ras invariably involved a GC----AT transition mutation of the first position of codon 12. Surprisingly, a Ki-ras gene that was activated by a GC----AT transition mutation at the same position was also detected in a single transformed bladder urothelial cell line derived from control cultures of mouse bladder cells. Together, our results indicate that Ki-ras activation in the DMBA-transformed bladder cell lines may not be a direct consequence of interaction of activated DMBA metabolites with the Ki-ras gene.

Mutation in mammalian cells by isomers of 5-methylchrysene diolepoxide.

The two pairs of diastereomeric anti- and syn-diolepoxide derivatives of 5-methylchrysene in both bay regions were tested for cytotoxicity and for mutagenicity at the hprt locus of chinese hamster V79 cells as determined by the ability of the cells to form colonies in medium containing 6-thioguanine. The concentration of compound in the cell media required to achieve 37% survival ranged from 0.3 to 4.5 micrograms/ml. Although the mutagenic effectiveness, i.e. the induced mutation frequency per unit concentration of compounds, varied over a 30-fold range, the mutagenic efficiency, i.e. the induced mutation frequency at an equivalent level of cell survival, showed only a 3-fold variation. The anti-1,2-diol-3,4-epoxide isomer (anti-5MCDE-I) was found to be the most mutagenic of the 5-methylchrysene diolepoxide isomers. This finding is consistent with previous observations on the tumorigenicity of these diolepoxides.