Selective phosphorylation of a nuclear envelope polypeptide by an endogenous protein kinase

Lam, Kit S.; Kasper, Charles B.

doi:10.1021/bi00569a012

Cited by 77 publications

(40 citation statements)

References 41 publications

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“…Recently, we have demonstrated that the direct addition of picomolar insulin to isolated rat liver nuclear envelopes stimulates NTPase activity (35), suggesting that insulin may regulate mRNA metabolism by means of this enzyme. Several groups have reported the presence of protein kinase and phosphatase activity in isolated nuclear envelopes (36)(37)(38), and it has been proposed that these enzymes may have a regulatory role in nucleocytoplasmic transport (36). Moreover, it has been suggested that phosphorylation reactions regulate nuclear envelope NTPase activity (39).…”

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confidence: 99%

Insulin regulation of protein phosphorylation in isolated rat liver nuclear envelopes: potential relationship to mRNA metabolism.

Purrello¹,

Burnham²,

Goldfine³

1983

Proc. Natl. Acad. Sci. U.S.A.

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The direct addition of insulin to highly purified nuclear envelopes prepared from the livers of diabetic rats resulted in a decrease in the incorporation of 32p into trichloroacetic acid-precipitable proteins. Autoradiography of 32P-labeled envelopes, solubilized in sodium dodecyl sulfate and subjected to electrophoresis, revealed that insulin decreased the phosphorylation of all major protein bands. Insulin produced detectable effects at concentrations between 0.1 and 1 pM, maximal effects at 10 pM, and progressively diminished effects at higher concentrations. Two insulin analogs, desdipeptide proinsulin and desoctapeptide insulin, had approximately 10% and 1%, respectively, the activity of native insulin. When nuclear envelopes were first phosphorylated with [y-32P]ATP and insulin was then added with an excess of unlabeled ATP, dephosphorylation was enhanced, suggesting that insulin was regulating nuclear envelope phosphatase activity. The direct addition of insulin to isolated rat liver nuclei in the presence of ATP stimulated the release of previously '4C-labeled trichloroacetic acid-precipitable mRNA-like material, and the direct addition of insulin to nuclear envelopes stimulated the activity of nucleoside triphosphatase, the enzyme that participates in mRNA nucleocytoplasmic transport. Moreover, the dose-response curves for these functions mirrored insulin's inhibition of nuclear envelope phosphorylation. These data suggest, therefore, a mechanism whereby insulin directly inhibits the phosphorylation of the nuclear envelope, leading in turn to the regulation of mRNA metabolism.Insulin has numerous effects on target cells, including regulation of membrane transport, enzyme activities, and DNA and RNA metabolism (1, 2). There is evidence that insulin, like other hormones, regulates a number of cellular functions by phosphorylation and dephosphorylation reactions (3-6). Moreover, effects of insulin on phosphorylation and dephosphorylation have recently been observed in broken cell preparations (7-11).We and others have demonstrated specific high-affinity binding sites for insulin on nuclei and nuclear envelopes of liver and other tissues (12)(13)(14)(15)(16)(17). Moreover, morphological studies have suggested that insulin can enter intact cells and interact with the nuclear envelope (18)(19)(20). However, the exact role of insulin in nuclear functions is unknown. There are a number of reports demonstrating that insulin increases mRNA levels in liver and other tissues (21)(22)(23)(24)(25)(26)(27)(28)(29). Further, it has recently been reported that the direct addition of insulin to isolated nuclei stimulates the release of mRNA (30). These observations suggest, therefore, that one action of insulin on the nucleus is to regulate mRNA metabolism.Several lines of evidence indicate that transport of mRNA from the nucleus is mediated by a specific enzyme of the nuclear envelope, the nuclear envelope nucleoside triphosphatase (NTPase; EC 3.6-. (39). Accordingly, in the present study we have investigated ...

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confidence: 99%

Insulin regulation of protein phosphorylation in isolated rat liver nuclear envelopes: potential relationship to mRNA metabolism.

Purrello¹,

Burnham²,

Goldfine³

1983

Proc. Natl. Acad. Sci. U.S.A.

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“…It also has been claimed that these two membrane structures are so similar as to be practically identical (5,6). But, some proteins have been reported to be specific for the nuclear envelope (13) and the induction of cytochrome P-450s by phenobarbital (PB) and 3-methylcholanthrene (MC) differs in the two membranes (3,11). In addition, the specific enzymatic activities of the nuclear envelope are usually lower than those of the ER (9,10,18,22).…”

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Immunoelectron microscopy of the outer membrane of rat hepatocyte nuclear envelopes in relation to the rough endoplasmic reticulum.

Matsuura

Masuda

Sakai

et al. 1983

Cell Struct. Funct.

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“…It remains insoluble in the course of the isolation procedure, which includes a treatment with detergent at low ionic strength, digestion of the nucleic acids, and extraction with a high-salt solution. Furthermore, it is at least partly resistant to 4 M urea. The presence of a protein kinase activity has been previously reported in isolated nuclear envelopes (26,28,29), but the phosphorylated products were not identified. In contrast with our results, this activity was found to be sensitive to nonionic detergents (28).…”

Section: Discussionmentioning

confidence: 85%

Protein kinase activity associated with the nuclear lamina.

Dessev

Iovcheva

Tasheva

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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A nuclear lamina-enriched fraction from Ehrlich ascites tumor cells contains a tightly bound protein kinase activity, which phosphorylates in vitro the nuclear lamins, a 52-kilodalton protein, and several unknown minor components. The enzyme(s) is thermolabile, independent of Ca2+ and cAMP, and inhibited by quercetin. After treatment with 4 M urea it remains bound to the nuclear lamina in an active state, but it is irreversibly inactivated in 6 M urea. The lamin proteins are phosphorylated on serine residues. Their two-dimensional phosphopeptide maps show multiple phosphorlation sites and a considerable similarity to the phosphopeptide maps of lamins labeled in vivo. Photoaffinity labeling experiments revealed several polypeptide fractions in the nuclear lamina fraction that are candidates for the protein kinase(s).The nuclear lamina (NL) is a proteinaceous meshwork located at the inner surface of the nuclear membrane (1, 2). The major constituents of the NL are three polypeptides with molecular masses between 60 and 75 kDa, termed lamins A, B, and C (2). It is thought that the NL plays a role in maintaining the structural organization of both the nuclear envelope (3, 4) and chromatin (5, 6) in the interphase nucleus. It has also been shown that the lamins are members of the intermediate filament protein family (7-11).The nuclear lamins are phosphoproteins. During interphase they exhibit a relatively low level of phosphorylation of about 0.2 mol of phosphate per mol of protein (12). However, when the NL is disassembled in mitosis (3, 4) there is an increased phosphorylation of the three lamins (4, 12). Little is known about the nature, location, and regulation of the protein kinase(s) involved in both interphase and mitotic phosphorylation of the lamins.We have recently developed a method for the isolation of the NL from Ehrlich ascites tumor (EAT) cells (13,14 (Amersham) at 25 uCi/ml in DME otherwise free of phosphate or methionine, respectively, supplemented with 5% dialyzed fetal calf serum.The original method developed for the isolation of NL from EAT cells (13, 14) was modified as follows. One milliliter of packed cells was resuspended in 30 tnl of solution 1 (0.25 M sucrose/5 mM EDTA/5 mM Tris HCI, pH 7.5/0.5 mM phenylmethylsulfonyl fluoride), incubated at 0C for 5 min, and centrifuged at 600 x g for 5 min. The cells were treated once more with 30 ml of solution I and twice with 30 ml of solution 11 (0.25 M sucrose/0.1 mM EDTA/5 mM Tris-HCI, pH 7.5/0.5 mM phenylmethylsulfonyl fluoride). Each time the cells were resuspended (Vortex mixer), incubated for 5 min at 0C, and collected by centrifugation at 600 x g for 5 min. The pellet was then resuspended in 10 ml of solution II, mixed with 10 ml of aqueous 1% Nonidet P-40 (Sigma), layered over a 60-ml cushion of solution III (0.25 M sucrose/0.1 mM EDTA/5 mM Tris HCl, pH 7.0), and spun at 3500 x g for 15 min. The resulting semitransparent pellet was resuspended in 10 ml of solution III and incubated for 30 min at room temperature in the presence of DNa...

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Selective phosphorylation of a nuclear envelope polypeptide by an endogenous protein kinase

Cited by 77 publications

References 41 publications

Insulin regulation of protein phosphorylation in isolated rat liver nuclear envelopes: potential relationship to mRNA metabolism.

Insulin regulation of protein phosphorylation in isolated rat liver nuclear envelopes: potential relationship to mRNA metabolism.

Immunoelectron microscopy of the outer membrane of rat hepatocyte nuclear envelopes in relation to the rough endoplasmic reticulum.

Protein kinase activity associated with the nuclear lamina.

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