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Intracellular distribution of mammalian protein kinase A catalytic subunit altered by conserved Asn2 deamidation.

Pepperkok R, Hotz-Wagenblatt A, König N, Girod A, Bossemeyer D, Kinzel V - J. Cell Biol. (2000)

Bottom Line: A major charge variant representing about one third of the enzyme in striated muscle results from deamidation in vivo of the Asn2 residue at the conserved NH(2)-terminal sequence myrGly-Asn-Ala (Jedrzejewski, P.T., A.Under all circumstances, the N/C ratio obtained with the encoded Asn2 form was significantly higher than that with the deamidated, Asp2 form; i.e., the Asn2 form reached a larger nuclear concentration than the Asp2 form.The model character for other signaling proteins starting with myrGly-Asn is discussed.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, D-69012 Heidelberg, Germany.

ABSTRACT
The catalytic (C) subunit of protein kinase A functions both in the cytoplasm and the nucleus. A major charge variant representing about one third of the enzyme in striated muscle results from deamidation in vivo of the Asn2 residue at the conserved NH(2)-terminal sequence myrGly-Asn-Ala (Jedrzejewski, P.T., A. Girod, A. Tholey, N. König, S. Thullner, V. Kinzel, and D. Bossemeyer. 1998. Protein Sci. 7:457-469). Because of the increase of electronegativity by generation of Asp2, it is reminiscent of a myristoyl-electrostatic switch. To compare the intracellular distribution of the enzymes, both forms of porcine or bovine heart enzyme were microinjected into the cytoplasm of mouse NIH 3T3 cells after conjugation with fluorescein, rhodamine, or in unlabeled form. The nuclear/cytoplasmic fluorescence ratio (N/C) was analyzed in the presence of cAMP (in the case of unlabeled enzyme by antibodies). Under all circumstances, the N/C ratio obtained with the encoded Asn2 form was significantly higher than that with the deamidated, Asp2 form; i.e., the Asn2 form reached a larger nuclear concentration than the Asp2 form. Comparable data were obtained with a human cell line. The differential intracellular distribution of both enzyme forms is also reflected by functional data. It correlates with the degree of phosphorylation of the key serine in CREB family transcription factors in the nucleus. Microinjection of myristoylated recombinant bovine Calpha and the Asn2 deletion mutant of it yielded N/C ratios in the same range as encoded native enzymes. Thus, Asn2 seems to serve as a potential site for modulating electronegativity. The data indicate that the NH(2)-terminal domain of the PKA C-subunit contributes to the intracellular distribution of free enzyme, which can be altered by site-specific in vivo deamidation. The model character for other signaling proteins starting with myrGly-Asn is discussed.

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Localization of microinjected PKA C-subunit fraction A and B by immunofluorescence. Purified porcine C-subunit fractions A and B (4 mg/ml each) were injected with 5 mM cAMP (in the pipette) into the cytoplasm of NIH 3T3 cells. After injection, cells were incubated at 37°C for different periods (30 min shown here) before they were fixed and stained for C-subunit using a polyclonal rabbit antibody and corresponding rhodamine-conjugated secondary antibodies (see Materials and Methods). The anti-PKA antibody was diluted sufficiently that only microinjected cells showed a significant fluorescent staining specific for microinjected C-subunit. Asterisks indicate the nucleus of neighboring noninjected cells. The image contrast was adjusted to better visualize growth cones in injected cells (marked by arrows). Therefore, the nucleus in the cell injected with fraction B appears to be saturated. As in the other experiments, for the quantification of N/C values, the original images were used where the gray values in all the images were always below the saturation level. The mean N/C ratio ± SD determined in this experiment from >100 cells was 1.9 ± 0.2 for fraction A and 3.3 ± 0.5 for fraction B at 30 min. Bar, 20 μm.
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Figure 5: Localization of microinjected PKA C-subunit fraction A and B by immunofluorescence. Purified porcine C-subunit fractions A and B (4 mg/ml each) were injected with 5 mM cAMP (in the pipette) into the cytoplasm of NIH 3T3 cells. After injection, cells were incubated at 37°C for different periods (30 min shown here) before they were fixed and stained for C-subunit using a polyclonal rabbit antibody and corresponding rhodamine-conjugated secondary antibodies (see Materials and Methods). The anti-PKA antibody was diluted sufficiently that only microinjected cells showed a significant fluorescent staining specific for microinjected C-subunit. Asterisks indicate the nucleus of neighboring noninjected cells. The image contrast was adjusted to better visualize growth cones in injected cells (marked by arrows). Therefore, the nucleus in the cell injected with fraction B appears to be saturated. As in the other experiments, for the quantification of N/C values, the original images were used where the gray values in all the images were always below the saturation level. The mean N/C ratio ± SD determined in this experiment from >100 cells was 1.9 ± 0.2 for fraction A and 3.3 ± 0.5 for fraction B at 30 min. Bar, 20 μm.

Mentions: Purified fractions A or B (both at 4 mg/ml) were microinjected into the cytoplasm of NIH 3T3 fibroblasts. They were incubated for 30 min at 37°C, fixed with methanol, incubated with affinity-purified antibodies, and stained with the fluorescent secondary antibody. As shown in Fig. 5, for the antibody dilutions used, the microinjected cells showed a strong fluorescent signal. In contrast, little fluorescence (<25% of the signal of injected cells) was obtained in noninjected cells (marked by stars in Fig. 5). Discrimination between injected and noninjected cells was facilitated by the increased appearance of growth cones (Fig. 5A and Fig. B, arrows) in cells injected with either fraction A or B. At time points longer than 30 min after microinjection, rounding up of the cells was observed (not shown). Similar morphological changes upon microinjection of the C-subunit into living cells have been reported by others (e.g., see Lamb et al. 1988, Lamb et al. 1989). They seem to indicate that the injected enzyme is functional. In all injected cells, a significant fluorescence signal was obtained in the cytoplasm and the nucleus. For both microinjected fraction A and B, an increased nuclear fluorescence was observed (Fig. 5).


Intracellular distribution of mammalian protein kinase A catalytic subunit altered by conserved Asn2 deamidation.

Pepperkok R, Hotz-Wagenblatt A, König N, Girod A, Bossemeyer D, Kinzel V - J. Cell Biol. (2000)

Localization of microinjected PKA C-subunit fraction A and B by immunofluorescence. Purified porcine C-subunit fractions A and B (4 mg/ml each) were injected with 5 mM cAMP (in the pipette) into the cytoplasm of NIH 3T3 cells. After injection, cells were incubated at 37°C for different periods (30 min shown here) before they were fixed and stained for C-subunit using a polyclonal rabbit antibody and corresponding rhodamine-conjugated secondary antibodies (see Materials and Methods). The anti-PKA antibody was diluted sufficiently that only microinjected cells showed a significant fluorescent staining specific for microinjected C-subunit. Asterisks indicate the nucleus of neighboring noninjected cells. The image contrast was adjusted to better visualize growth cones in injected cells (marked by arrows). Therefore, the nucleus in the cell injected with fraction B appears to be saturated. As in the other experiments, for the quantification of N/C values, the original images were used where the gray values in all the images were always below the saturation level. The mean N/C ratio ± SD determined in this experiment from >100 cells was 1.9 ± 0.2 for fraction A and 3.3 ± 0.5 for fraction B at 30 min. Bar, 20 μm.
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Figure 5: Localization of microinjected PKA C-subunit fraction A and B by immunofluorescence. Purified porcine C-subunit fractions A and B (4 mg/ml each) were injected with 5 mM cAMP (in the pipette) into the cytoplasm of NIH 3T3 cells. After injection, cells were incubated at 37°C for different periods (30 min shown here) before they were fixed and stained for C-subunit using a polyclonal rabbit antibody and corresponding rhodamine-conjugated secondary antibodies (see Materials and Methods). The anti-PKA antibody was diluted sufficiently that only microinjected cells showed a significant fluorescent staining specific for microinjected C-subunit. Asterisks indicate the nucleus of neighboring noninjected cells. The image contrast was adjusted to better visualize growth cones in injected cells (marked by arrows). Therefore, the nucleus in the cell injected with fraction B appears to be saturated. As in the other experiments, for the quantification of N/C values, the original images were used where the gray values in all the images were always below the saturation level. The mean N/C ratio ± SD determined in this experiment from >100 cells was 1.9 ± 0.2 for fraction A and 3.3 ± 0.5 for fraction B at 30 min. Bar, 20 μm.
Mentions: Purified fractions A or B (both at 4 mg/ml) were microinjected into the cytoplasm of NIH 3T3 fibroblasts. They were incubated for 30 min at 37°C, fixed with methanol, incubated with affinity-purified antibodies, and stained with the fluorescent secondary antibody. As shown in Fig. 5, for the antibody dilutions used, the microinjected cells showed a strong fluorescent signal. In contrast, little fluorescence (<25% of the signal of injected cells) was obtained in noninjected cells (marked by stars in Fig. 5). Discrimination between injected and noninjected cells was facilitated by the increased appearance of growth cones (Fig. 5A and Fig. B, arrows) in cells injected with either fraction A or B. At time points longer than 30 min after microinjection, rounding up of the cells was observed (not shown). Similar morphological changes upon microinjection of the C-subunit into living cells have been reported by others (e.g., see Lamb et al. 1988, Lamb et al. 1989). They seem to indicate that the injected enzyme is functional. In all injected cells, a significant fluorescence signal was obtained in the cytoplasm and the nucleus. For both microinjected fraction A and B, an increased nuclear fluorescence was observed (Fig. 5).

Bottom Line: A major charge variant representing about one third of the enzyme in striated muscle results from deamidation in vivo of the Asn2 residue at the conserved NH(2)-terminal sequence myrGly-Asn-Ala (Jedrzejewski, P.T., A.Under all circumstances, the N/C ratio obtained with the encoded Asn2 form was significantly higher than that with the deamidated, Asp2 form; i.e., the Asn2 form reached a larger nuclear concentration than the Asp2 form.The model character for other signaling proteins starting with myrGly-Asn is discussed.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, D-69012 Heidelberg, Germany.

ABSTRACT
The catalytic (C) subunit of protein kinase A functions both in the cytoplasm and the nucleus. A major charge variant representing about one third of the enzyme in striated muscle results from deamidation in vivo of the Asn2 residue at the conserved NH(2)-terminal sequence myrGly-Asn-Ala (Jedrzejewski, P.T., A. Girod, A. Tholey, N. König, S. Thullner, V. Kinzel, and D. Bossemeyer. 1998. Protein Sci. 7:457-469). Because of the increase of electronegativity by generation of Asp2, it is reminiscent of a myristoyl-electrostatic switch. To compare the intracellular distribution of the enzymes, both forms of porcine or bovine heart enzyme were microinjected into the cytoplasm of mouse NIH 3T3 cells after conjugation with fluorescein, rhodamine, or in unlabeled form. The nuclear/cytoplasmic fluorescence ratio (N/C) was analyzed in the presence of cAMP (in the case of unlabeled enzyme by antibodies). Under all circumstances, the N/C ratio obtained with the encoded Asn2 form was significantly higher than that with the deamidated, Asp2 form; i.e., the Asn2 form reached a larger nuclear concentration than the Asp2 form. Comparable data were obtained with a human cell line. The differential intracellular distribution of both enzyme forms is also reflected by functional data. It correlates with the degree of phosphorylation of the key serine in CREB family transcription factors in the nucleus. Microinjection of myristoylated recombinant bovine Calpha and the Asn2 deletion mutant of it yielded N/C ratios in the same range as encoded native enzymes. Thus, Asn2 seems to serve as a potential site for modulating electronegativity. The data indicate that the NH(2)-terminal domain of the PKA C-subunit contributes to the intracellular distribution of free enzyme, which can be altered by site-specific in vivo deamidation. The model character for other signaling proteins starting with myrGly-Asn is discussed.

Show MeSH
Related in: MedlinePlus