UV-induced hyperphosphorylation of replication protein a depends on DNA replication and expression of ATM protein.

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4-8 h) to UV radiation (10-30 J/m(2)). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

An erythropoietin fusion protein comprised of identical repeating domains exhibits enhanced biological properties.

The hematopoietic growth factor erythropoietin (Epo) initiates its intracellular signaling cascade by binding to and inducing the homodimerization of two identical receptor molecules. We have now constructed and expressed in COS cells a cDNA encoding a fusion protein consisting of two complete human Epo domains linked in tandem by a 17-amino acid flexible peptide. On SDS-polyacrylamide gel electrophoresis, the Epo-Epo fusion protein migrated as a broad band with an average apparent molecular mass of 76 kDa, slightly more than twice the average apparent molecular mass of Epo, 37 kDa. Enzymatic N-deglycosylation resulted in an Epo-Epo species that migrated on SDS-polyacrylamide gel electrophoresis as a narrow band with an average apparent molecular mass of 39 kDa. The specific activity of the Epo-Epo fusion protein in vitro (1,007 IU/microgram; 76 IU/pmol) was significantly greater than that of Epo (352 IU/microgram; 13 IU/pmol). Moreover, secretion of Epo-Epo by COS cells was 8-fold greater than that of Epo. Subcutaneous administration of a single dose of Epo-Epo to mice resulted in a significant increase in red blood cell production within 7 days. In contrast, administration of an equivalent dose of conventional recombinant Epo was without effect. The pharmacokinetic behavior of Epo-Epo differed significantly from that of Epo. The results suggest that Epo-Epo may have important biological and therapeutic advantages.

Role of N-myristylation in targeting of band 4.2 (pallidin) in nonerythroid cells.

Band 4.2 (pallidin) is a major erythrocyte membrane protein which has been detected in a number of nonerythroid cell types. Increasing evidence suggests that band 4.2 is involved in maintaining membrane stability in the erythrocyte. For example, band 4.2 binds to the integral membrane protein band 3 and to cytoskeletal proteins in the erythrocyte membrane, and band 4.2 deficiency results in varying degrees of hemolytic anemia. We have previously shown that human erythrocyte band 4.2 is myristylated at its penultimate glycine. Here we report that when expressed in both Sf9 and COS cells, myristylated forms of band 4.2 are detected at different intracellular locations than nonmyristylated forms. We also show that the unspliced form of human erythrocyte band 4.2 (a minor form in reticulocytes which contains an additional 30 amino acids after the first three N-terminal amino acids compared to the major erythroid form) is myristylated only at a barely detectable level, while mouse erythrocyte band 4.2 (homologous to the major erythroid form of human band 4.2) is myristylated at a level comparable to that of human band 4.2. These results suggest that myristylation plays a key role in the targeting of band 4.2 to specific intracellular locations and is likely to have a role in the function of this protein.

Human erythrocyte protein 4.2, a high copy number membrane protein, is N-myristylated.

Band 4.2 is a major protein of the erythrocyte membrane which has been immunologically detected in a variety of cell types and is apparently essential for normal erythrocyte membrane function. Since band 4.2 has unusual solubility and membrane binding properties and has an N-terminal glycine following the initiating methionine, we explored the possibility that band 4.2 is myristylated. When Sf9 cells infected with a recombinant band 4.2 Baculovirus were incubated with [3H]myristic acid, label became incorporated into recombinant band 4.2 protein and resisted extraction with hydroxylamine. Consistent with N-terminal myristylation, the incorporation of label was dependent upon protein synthesis. The fatty acid covalently bound to recombinant band 4.2 was definitively identified as myristic acid by recovering the fatty acid after hydrolysis of band 4.2 and examining its migration relative to standards in thin layer chromatography. It was determined that native erythrocyte band 4.2 is an N-myristylated protein by reverse phase high performance liquid chromatography detection of an azlactone derivative of N-myristylglycine after mild acid hydrolysis and azlactone derivatization of the purified protein. Study of myristylation of band 4.2, an abundant normal cellular protein, and its role in membrane binding may produce insights relevant to other myristylated cellular proteins.

Studies on proteins that co-purify with smooth muscle vinculin: identification of immunologically related species in focal adhesions of nonmuscle and Z-lines of muscle cells.

Membrane extracts from chicken smooth muscle contain, along with filamin, vinculin and alpha actinin, a group of polypeptides that have the ability to interact with the "barbed end" of actin filaments. These low molecular mass polypeptides were designated as HA1 (Wilkins, J.A., and S. Lin, 1986, J. Cell Biol., 102:1085-1092). In this study, polyclonal antibodies raised against the HA1 preparation were used to study the cellular localization and tissue distribution of these polypeptides. Immunofluorescence experiments revealed a primary localization of staining at the ends of stress fibers on the ventral surface of cultured chicken embryo fibroblasts, i.e., those areas known as the focal adhesions. Specific staining was also seen at the Z-lines of both skeletal muscle myofibrils and cultured embryonic heart cells. Immunoblotting analyses of proteins from different tissues prepared to avoid proteolytic degradation showed a much different pattern than that of HA1 itself. Immunoreactive polypeptides with reduced molecular masses of 200,000 and 150,000 D were found in smooth muscle and fibroblasts while 200 and 60 kD polypeptides were found in cardiac muscle tissue. The antibodies recognized 60- and 31-kD polypeptides on immunoblots of chicken breast muscle. The results from this study strongly suggest that the polypeptides in HA1 arose from proteolysis of high molecular mass molecules. The studies also raise the possibility that immunologically related proteins in muscle and nonmuscle cells may be involved in linking actin filaments to Z-lines and membranes, respectively.

Actin filament organization of the Dunning R3327 rat prostatic adenocarcinoma system: correlation with metastatic potential.

Recently, Volk, Geiger, and Raz (Cancer Res., 44: 811-824, 1984) addressed the question of whether variations in actin organization in clones of the murine K-1735 melanoma tumor correlated with their metastatic capability. Using immunofluorescence techniques, they found that clones which had a more ordered actin network were less metastatic, whereas clones having a diffuse actin staining pattern were more metastatic. Similarly, we have found that in the Dunning rat R3327 prostatic adenocarcinoma tumor system, the non-metastatic (less than 0.1%) H-prostatic tumor cell line has a prominent network of actin filament bundles, whereas the highly metastatic (greater than 90%) MatLyLu cell line has a diffuse actin staining pattern. In the low-metastatic (less than 10%) AT1 cell line an intermediate actin organization between H and MatLyLu was observed. Analysis of cell extracts from H- and MatLyLu-cells revealed differences in the level of activity of cellular proteins which affect actin filament assembly and structure in a manner similar to that of the cytochalasins, fungal metabolites which bind with high affinity to the fast-growing end of actin filaments. Extracts of MatLyLu were significantly more effective than those of H-cells in decreasing the extent of actin filament network formation and in inhibiting the rate of filament assembly by blocking monomer addition onto the fast-growing end. Measurements of spin-lattice nuclear magnetic resonance water proton relaxation times (T1) were made in surgically removed tumor tissue from four sublines (H, AT1, MatLyLu, and MatLu) of the Dunning R3327 tumor system. The highly metastatic cell lines had significantly longer water proton T1 relaxation times than did the lines with low metastatic potential. These differences in T1 may reflect the observed alterations in organization of actin filaments within these various sublines of the Dunning R3327 prostatic adenocarcinoma tumor system.

Interaction of filipin with junctional membrane at different stages of the junction's life history.

The relationship of filipin-sterol complexes to tight and gap junctions during their formation, maturation, internalization, and degradation was studied in separate cell lines. Filipin-sterol complexes tended to be excluded from mature junctions in tight junction forming COLO 316 cells and gap junction forming SW-13 cells. Once internalized, unlabeled junctional membrane appeared to fuse with heavily labeled vesicles, presumably lysosomes. Although the absence of filipin-sterol complexes from junctional membrane does not necessarily reflect the absolute sterol content of this membrane, the fact that filipin-sterol complexes are largely excluded from these areas indicates that this membrane is different from surrounding membrane. The absence of filipin-sterol complexes also permits the visualization of 'mixing' of this specialized unlabeled membrane domain with other filipin labeled membrane systems.

Endocytosis of cell-cell junctions and spontaneous cell disaggregation in a cultured human ovarian adenocarcinoma. (COLO 316).

Although cultured COLO 316 human ovarian adenocarcinoma cells are joined by extensive tight junctions and numerous demosomes in confluent monolayers, viable cells may be spontaneously released into the nutrient medium. Intracytoplasmic vesicles containing tight junctional and desmosomal elements were identified in freeze-fracture and thin section preparations of the released cells and some vesicles exhibited structural signs of degradation. Possible mechanisms for tight junctional and desmosomal interiorization and the possible relationship between junctional interiorization and certain malignant behaviors are discussed.