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Nuclear-membrane-associated protein kinase and substrates. The effect of growth state on activity and specificity.

ABSTRACT: Nuclear membrane was isolated from cultured cells by two different techniques. The first technique employed sonication to lyse the nuclei, followed by treatment with citrate buffer to strip away the chromatin. The second procedure involved incubation with the polyanion heparin to lyse the nuclei. In both procedures, the nuclear membrane was purified by isopycnic centrifugation on discontinuous sucrose gradients. Both preparations contained endogenous protein kinase activity and phosphorylated endogenous membrane proteins. The phosphoprotein profiles and characteristics of the phosphorylation reaction were very similar for the two preparations, except that the heparin-prepared membranes lacked two major phosphorpoteins which were present in membranes prepared by sonication. The growth state of the culture had a dramatic effect on nuclear-membrane protein phosphorylation. Proliferating cells exhibited a 3-5-fold greater extent of phosphorylation of nuclear-membrane proteins than did quiescent cells. The increased phosphorylation observed in proliferating cells implies that regulation at the level of the nuclear membrane may be an important site for regulation of cell-cycle events.

SUBMITTER: Kletzien RF

PROVIDER: S-EPMC1163107 | biostudies-other | 1981 Jun

REPOSITORIES: biostudies-other

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Project description:The nuclear envelope (NE) consists of two concentric nuclear membranes separated by the lumen, an ∼40-nm-wide fluid layer. NE proteins are implicated in important cellular processes ranging from gene expression to nuclear positioning. Although recent progress has been achieved in quantifying the assembly states of NE proteins in their native environment with fluorescence fluctuation spectroscopy, these studies raised questions regarding the association of NE proteins with nuclear membranes during the assembly process. Monitoring the interaction of proteins with membranes is important because the binding event is often associated with conformational changes that are critical to cellular signaling pathways. Unfortunately, the close physical proximity of both membranes poses a severe experimental challenge in distinguishing luminal and membrane-associated NE proteins. This study seeks to address this problem by introducing new, to our knowledge, fluorescence-based assays that overcome the restrictions imposed by the NE environment. We found that luminal proteins violate the Stokes-Einstein relation, which eliminates a straightforward use of protein mobility as a marker of membrane association within the NE. However, a surprising anomaly in the temperature-dependent mobility of luminal proteins was observed, which was developed into an assay for distinguishing between soluble and membrane-bound NE proteins. We further introduced a second independent tool for distinguishing both protein populations by harnessing the previously reported undulations of the nuclear membranes. These membrane undulations introduce local volume changes that produce an additional fluorescence fluctuation signal for luminal, but not for membrane-bound, proteins. After testing both methods using simple model systems, we apply the two assays to investigate a previously proposed model of membrane association for the luminal domain of SUN2, a constituent protein of the linker of nucleoskeleton and cytoskeleton complex. Finally, we investigate the effect of C- and N-terminal tagging of the luminal ATPase torsinA on its ability to associate with nuclear membranes.

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Nuclear-membrane-associated protein kinase and substrates. The effect of growth state on activity and specificity.

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