scholarly journals Movement of the free catalytic subunit of cAMP-dependent protein kinase into and out of the nucleus can be explained by diffusion.

1993 ◽  
Vol 4 (10) ◽  
pp. 993-1002 ◽  
Author(s):  
A T Harootunian ◽  
S R Adams ◽  
W Wen ◽  
J L Meinkoth ◽  
S S Taylor ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Intracellular Camp ◽  
Dependent Protein Kinase ◽  
Regulatory Subunits ◽  
Camp Dependent Protein Kinase ◽  
Block Movement ◽  
C Subunit ◽  
Dependent Protein

The catalytic (C) subunit of cyclic AMP (cAMP) dependent protein kinase (PKA) has previously been shown to enter and exit the nucleus of cells when intracellular cAMP is raised and lowered, respectively. To determine the mechanism of nuclear translocation, fluorescently labeled C subunit was injected into living REF52 fibroblasts either as free C subunit or in the form of holoenzyme (PKA) in which the catalytic and regulatory subunits were labeled with fluorescein and rhodamine, respectively. Quantification of nuclear and cytoplasmic fluorescence intensities revealed that free C subunit nuclear accumulation was most similar to that of macromolecules that diffuse into the nucleus. A glutathione S-transferase-C subunit fusion protein did not enter the nucleus following cytoplasmic microinjection. Puncturing the nuclear membrane did not decrease the nuclear concentration of C subunit, and C subunit entry into the nucleus did not appear to be saturable. Cooling or depleting cells of energy failed to block movement of C subunit into the nucleus. Photobleaching experiments showed that even after reaching equilibrium at high [cAMP], individual molecules of C subunit continued to leave the nucleus at approximately the same rate that they had originally entered. These results indicate that diffusion is sufficient to explain most aspects of C subunit subcellular localization.

scholarly journals Cyclic AMP-dependent protein kinase in rat mammary tissue: expression of catalytic and regulatory subunits throughout pregnancy and lactation

1994 ◽  
Vol 301 (3) ◽  
pp. 807-812 ◽  
Author(s):  
R A Gardner ◽  
M T Travers ◽  
M C Barber ◽  
W R Miller ◽  
R A Clegg
Keyword(s):  
Catalytic Activity ◽  
Protein Kinase ◽  
Mammary Development ◽  
Mammary Tissue ◽  
Early Lactation ◽  
Dependent Protein Kinase ◽  
Regulatory Subunits ◽  
C Subunit ◽  
Pregnancy And Lactation ◽  
Dependent Protein

‘Expressed’ and ‘total’ activities of cyclic AMP-dependent protein kinase (PK-A) were measured in extracts of rat mammary tissue sampled throughout pregnancy and lactation. Expression of the genes encoding the catalytic subunit (C-subunit) isoforms C alpha and C beta was examined by Northern blotting, as a function of mammary development, to determine relative levels of their respective mRNAs. The content of C-subunit protein (all isoforms) was estimated immunochemically and related to levels of C-subunit catalytic activity and of mRNAs. It was found that C-subunit isoform mRNAs are expressed co-ordinately during mammary development and that a marked decline in expression, per cell, at around parturition is paralleled by a fall in ‘total’ PK-A activity. The ‘expressed’ activity of PK-A activity underwent characteristic changes throughout pregnancy and lactation, reaching a peak late in pregnancy. The PK-A activity ratio reached a peak in early lactation. C-subunit protein mass closely parallel ‘total’ PK-A activity throughout pregnancy and lactation, thereby demonstrating the constancy of C-subunit specific catalytic activity during these developmental events. Regulatory subunits (R-subunits) were probed with the photoaffinity label 8-azido-[32P]cAMP. The abundance of R-II as a proportion of total R-subunit increased throughout pregnancy and lactation, and quantitative analysis of the photoaffinity labelling suggested inconstancy in the ratio of R:C subunits, with highest values occurring in late pregnancy/early lactation.

scholarly journals Localization of nuclear subunits of cyclic AMP-dependent protein kinase by the immunocolloidal gold method.

1985 ◽  
Vol 101 (3) ◽  
pp. 965-975 ◽  
Author(s):  
M R Kuettel ◽  
S P Squinto ◽  
J Kwast-Welfeld ◽  
G Schwoch ◽  
J S Schweppe ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Protein Kinase ◽  
Hepatoma Cells ◽  
Catalytic Subunit ◽  
Immunogold Electron Microscopy ◽  
Dependent Protein Kinase ◽  
Regulatory Subunits ◽  
Camp Dependent Protein Kinase ◽  
Specific Antisera ◽  
Dependent Protein

An immunocolloidal gold electron microscopy method is described allowing the ultrastructural localization and quantitation of the regulatory subunits RI and RII and the catalytic subunit C of cAMP-dependent protein kinase. Using a postembedding indirect immunogold labeling procedure that employs specific antisera, the catalytic and regulatory subunits were localized in electron-dense regions of the nucleus and in cytoplasmic areas with a minimum of nonspecific staining. Antigenic domains were localized in regions of the heterochromatin, nucleolus, interchromatin granules, and in the endoplasmic reticulum of different cell types, such as rat hepatocytes, ovarian granulosa cells, and spermatogonia, as well as cultured H4IIE hepatoma cells. Morphometric quantitation of the relative staining density of nuclear antigens indicated a marked modulation of the number of subunits per unit area under various physiologic conditions. For instance, following partial hepatectomy in rats, the staining density of the nuclear RI and C subunits was markedly increased 16 h after surgery. Glucagon treatment of rats increased the staining density of only the nuclear catalytic subunit. Dibutyryl cAMP treatment of H4IIE hepatoma cells led to a marked increase in the nuclear staining density of all three subunits of cAMP-dependent protein kinase. These studies demonstrate that specific antisera against cAMP-dependent protein kinase subunits may be used in combination with immunogold electron microscopy to identify the ultrastructural location of the subunits and to provide a semi-quantitative estimate of their relative cellular density.

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