Nuclear localization of aldolase A in pig cardiomyocytes

The subcellular localization of the muscle aldolase (aldolase A) in cardiomyocytes was determined immunocytochemically by light and electron microscopy. The enzyme was localized in the cytoplasm and also in cardiomyocyte nuclei. Inside the nuclei it was preferentially localized in the heterochromatin region. The nuclear localization was confirmed by the measurement of aldolase activity in subcellular fractions of a heart muscle, and in isolated nuclei of cardiomyocytes. There was no detectable aldolase activity in isolated cardiomyocyte nuclei fractions if the fraction was not preincubated with a solution containing Triton X-100 and KCl. The calculated concentration of aldolase in the nucleus was about 0.6 micro M. This paper is the first report on the localization of aldolase A inside cardiomyocyte nuclei.     

Nuclear localization signal receptor affinity correlates with in vivo localization in Saccharomyces cerevisiae

Nuclear localization signals (NLSs) target proteins into the nucleus through mediating interactions with nuclear import receptors. Here, we perform a quantitative analysis of the correlation between NLS receptor affinity and the steady-state distribution of NLS-bearing cargo proteins between the cytoplasm and the nucleus of live yeast, which reflects the relative import rates of various NLS sequences. We find that there is a complicated, but monotonic quantitative relationship between the affinity of an NLS for the import receptor, importin alpha, and the steady-state accumulation of the cargo in the nucleus. This analysis takes into consideration the impact of protein size. In addition, the hypothetical upper limit to an NLS affinity for the receptors is explored through genetic approaches. Overall, our results indicate that there is a correlation between the binding affinity of an NLS cargo for the NLS receptor, importin alpha, and the import rate for this cargo. This correlation, however, is not maintained for cargoes that bind to the NLS receptor with very weak or very strong affinity.     

Cardiolipin composition correlates with prostate cancer cell proliferation

Molecular and Cellular Biochemistry - Prostate cancer (PC) is the second most diagnosed cancer in men. It has been recognized that diet can play a crucial role in PC genesis and progression. In...     

Subcellular localization of aldolase B

The localization of the aldolase B isozyme was determined immunohistochemically in rat kidney and liver using a polyclonal antibody. Aldolase B was preferentially localized in a nuclear region of hepatocytes from the periportal region and was absent in those from the perivenous region. Aldolase B was also preferentially localized in the proximal tubules and was absent in other structures of the renal cortex as well as in the renal medulla. Using reflection confocal microscopy, the enzyme was preferentially localized in a nuclear position in liver and renal cells, which was similar to the cellular and intracellular location found for the gluconeogenic enzyme fructose-1,6-bisphosphatase (Sáez et al. [1996] J. Cell. Biochem. 63:453-462). Subcellular fractionation studies followed by enzyme activity assays revealed that aldolase activity was associated with subcellular particulate structures. Overall, the data suggest that different aldolase isoenzymes are needed in the glycolytic and gluconeogenic pathways.     

Nuclear localization of the titin Z1Z2Zr domain and role in regulating cell proliferation

Titin (also called connectin) is a major protein in sarcomere assembly as well as providing elastic return of the sarcomere postcontraction in cardiac and striated skeletal muscle tissues. In addition, it has been speculated that titin is associated with nuclear functions, including chromosome and spindle formation, and regulation of muscle gene expression. In the present study, a short isoform of titin was detected in a human osteoblastic cell line, MG-63 cells, by both immunostaining and Western blot analysis. Confocal images of titin staining showed both cytoplasmic and nuclear localization in a punctate pattern. Therefore, we hypothesized that human titin may contain a nuclear localization signal (NLS). A functional NLS, 200-PAKKTKT-206, located in a low-complexity, titin-specific region between Z2 and Z repeats, was found by sequentially deleting segments of the NH₂-terminal sequence in conjunction with an enhanced green fluorescent protein reporter system and confirmed by site-directed mutagenesis. Overexpression of titin's amino terminal fragment (Z1Z2Zr) in human osteoblasts (MG-63) increased cell proliferation by activating the Wnt/β-catenin pathway. RT-PCR screens of tissue panels demonstrated that residues 1–206 were ubiquitously expressed at low levels in all tissues and cell types analyzed. Our data implicate a dual role for titin's amino terminal region, i.e., a novel nuclear function promoting cell division in addition to its known structural role in Z-line assembly. Wnt; catenin; osteoblast     

Negative charge correlates with neural expression in vertebrate aldolase isozymes

Electrophoretic studies suggest that negatively charged neural proteins are a general feature of jawed vertebrates. In an apparent example of this, teleost fish express three aldolase isozymes, one of which is expressed predominantly in neural tissues and is more negatively charged than its more generally expressed paralogues. We characterized three aldolase isozymes from a single species of teleost fish, zebrafish (Danio rerio). These sequences indicated that the correlation of net negative charge and neural expression suggested in other species by gel electrophoresis was supported by sequence analysis. When aldolase sequences from the databases were included in phylogenetic analyses, the negative charge/neural expression phenomenon was observed across the gnathostome vertebrate sequences examined. We found no evidence for a period of positive Darwinian selection resulting in an accumulation of negatively charged amino acids during the evolution of the neural aldolase isozymes. This is likely attributable, however, to limitations associated with the age of the duplication responsible for the neural isozyme and the reconstruction of ancestral sequences.     

Nuclear and cytoplasmic localization of neural stem cell microRNAs

Although generally regarded as functional in the cytoplasm, a number of microRNAs (miRNAs) have been found in the nucleus, possibly with a role in gene regulation. Here we report that, in fact, a substantial fraction of all human miRNAs are present in the nucleus of neural stem cells. Further, subsets of these miRNAs display consistently higher standardized rank in the nucleus than in the cytoplasm of these cells, as identified with an RT-qPCR technology and confirmed by microarray analysis. Likewise, other miRNAs display higher cytoplasmic standardized ranks. Three samples were partitioned into nuclear and cytoplasmic fractions in six assays for 373 miRNAs. From the 100 most highly expressed miRNAs, standard scores of nuclear and cytoplasmic concentrations were determined. Among those, 21 miRNAs had all three nuclear standard scores higher than all three cytoplasmic scores; likewise, 31 miRNAs had consistently higher cytoplasmic scores. Random concentrations would result in only five in each set. Remarkably, if one miRNA has a high standard score in a compartment, then other miRNAs having the same 5' seeds and certain similar 3' end patterns are also highly scored in the same way. That is, in addition to the seed sequence, 3' sequence similarity criteria identify families of mature miRNAs with consistently high nuclear or cytoplasmic expression.     

Autocrine-mediated activation of STAT3 correlates with cell proliferation in breast carcinoma lines

The intracellular signals driving the proliferation of breast carcinoma (BC) cells have been widely studied. Both the mitotic and metastatic potential of BC cells have been linked to the frequent overexpression of ErbB family members. Other signaling molecules, including the estrogen receptor, the tyrosine kinases c-Src and Syk, and STAT proteins, especially STAT3, have also been implicated in BC tumor growth. Here we have examined ErbB and STAT protein expression and activation in six BC-derived cell lines. ErbB expression and tyrosine phosphorylation varied considerably among the six cell lines. However, STAT protein expression and activation were more consistent. Two levels of STAT3 activation were distinguished in DNA-binding assays: an epidermal growth factor-inducible, high level that requires both ErbB1 and Janus kinase (JAK) activity and an elevated serum-dependent level that is maintained by autocrine/paracrine signaling and requires JAK activity but is independent of ErbB1 kinase activity. BC cell growth could be inhibited by dominant-negative versions of STAT3 and the JAK inhibitor AG490 but not by PD153035 or PD168393, inhibitors of ErbB1 kinase activity. This indicates that BC cell proliferation may be a consequence of STAT3 activation by autocrine/paracrine signals.     

Intracellular localization of fructose 1,6-bisphosphate aldolase.

Submission of a rat liver homogenate made in 250 mM sucrose-1 mM EDTA to centrifugation between 9,500 times g for 10 min and 105,000 times g for 60 min results in the sedimentation of 60 to 70% of the total cellular fructose 1,6-bisphosphate aldolase (EC 4.1.2.13). Under these conditions only about one-quarter of the total triose phosphate dehydrogenase and phosphoglycerate kinase appears in the microsomal fraction. Ultrastructural immunologic localization techniques have demonstrated that the aldolase is associated with the endoplasmic reticulum, in situ. The binding of this enzyme to the membrane is sensitive to changes in pH with an optimum at 6.0, and to increasing concentrations of NaCl and fructose 1,6-bisphosphate, being about 100-fold more sensitive to the ester than to the inorganic salt.     

Nuclear/cytoplasmic localization of Akt activity in the cell cycle

Summary. The serine/threonine protein kinase Akt (also known as PKB) is a proto-oncogene and one of the most frequently hyperactivated kinases in human cancer. Its activation downstream of growth-factor-stimulated phosphatidylinositide-3′-OH kinase activity plays a role in the control of cell cycle, cell growth, apoptosis and cell energy metabolism. Akt phosphorylates some thousand downstream substrates, including typical cytoplasmic as well as nuclear proteins. Accordingly, it is not surprising that Akt activity can be found in both, the cytoplasm and the nucleus. Here we report the cell cycle regulation of nuclear and cytoplasmic Akt activity in mammalian cells. These data provide new insights into the regulation of Akt activity and have implications for future studies on the regulation of the wide variety of different nuclear and cytoplasmic Akt substrates.     

Pendulin, a Drosophila protein with cell cycle-dependent nuclear localization, is required for normal cell proliferation

We describe the dynamic intracellular localization of Drosophila Pendulin and its role in the control of cell proliferation. Pendulin is a new member of a superfamily of proteins which contains Armadillo (Arm) repeats and displays extensive sequence similarities with the Srp1 protein from yeast, with RAG-1 interacting proteins from humans, and with the importin protein from Xenopus. Almost the entire polypeptide chain of Pendulin is composed of degenerate tandem repeats of approximately 42 amino acids each. A short NH2-terminal domain contains adjacent consensus sequences for nuclear localization and cdc2 kinase phosphorylation. The subcellular distribution of Pendulin is dependent on the phase of cell cycle. During interphase, Pendulin protein is exclusively found in the cytoplasm of embryonic cells. At the transition between G2 and M-phase, Pendulin rapidly translocates into the nuclei where it is distributed throughout the nucleoplasm and the areas around the chromosomes. In the larval CNS, Pendulin is predominantly expressed in the dividing neuroblasts, where it undergoes the same cell cycle-dependent redistribution as in embryos. Pendulin is encoded by the oho31 locus and is expressed both maternally and zygotically. We describe the phenotypes of recessive lethal mutations in the oho31 gene that result in a massive decrease or loss of zygotic Pendulin expression. Hematopoietic cells of mutant larvae overproliferate and form melanotic tumors, suggesting that Pendulin normally acts as a blood cell tumor suppressor. In contrast, growth and proliferation in imaginal tissues are reduced and irregular, resulting in abnormal development of imaginal discs and the CNS of the larvae. This phenotype shows that Pendulin is required for normal growth regulation. Based on the structure of the protein, we propose that Pendulin may serve as an adaptor molecule to form complexes with other proteins. The sequence similarity with importin indicates that Pendulin may play a role in the nuclear import of karyophilic proteins and some of these may be required for the normal transmission and function of proliferative signals in the cells.     

Interaction of aldolase A with lecithin liposomes

The aldolase A binding to the lecithin liposomes (Kd = 2.4 +/- 0.1 X 10(-3) M) has been shown by the fluorescence and tryptophan phosphorescence at the room temperature. The interaction is accompanied by an increase in the phospholipid bilayer microviscosity, and some conformational changes in the hydrophobic part of the enzyme, pronouncing themselves in Trp-147 environment rigidity, decrease. The observation of membrane viscosity vs. incubation time revealed practically instant enzyme-membrane interaction and no gradual incorporation. The accessibility of the NAD-binding domain of aldolase for NADH in the liposome presence remains unaltered.