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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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Decay of microinjected PKA C-subunit fraction A and B. NIH 3T3 cells were microinjected with purified PKA C-subunit fraction A or B at 4 mg/ml. At different time points after injection, cells were fixed and stained by immunofluorescence for injected PKA and subsequently mounted on glass slides. Images of injected cells were acquired as described in Materials and Methods. The average PKA-specific fluorescence intensity (including the nucleus and cytoplasm) in injected cells was determined, and the value obtained for cells fixed immediately after injection (t = 0) was set at 100%. The averages and SDs of two independent experiments with at least 65 cells measured are shown. Degradation of the injected PKA proteins appeared to be similar for the fraction A and B.
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Figure 7: Decay of microinjected PKA C-subunit fraction A and B. NIH 3T3 cells were microinjected with purified PKA C-subunit fraction A or B at 4 mg/ml. At different time points after injection, cells were fixed and stained by immunofluorescence for injected PKA and subsequently mounted on glass slides. Images of injected cells were acquired as described in Materials and Methods. The average PKA-specific fluorescence intensity (including the nucleus and cytoplasm) in injected cells was determined, and the value obtained for cells fixed immediately after injection (t = 0) was set at 100%. The averages and SDs of two independent experiments with at least 65 cells measured are shown. Degradation of the injected PKA proteins appeared to be similar for the fraction A and B.

Mentions: To evaluate the possibility that the apparent differences of the N/C ratios obtained with the fractions A and B were caused by a preferential degradation of one of the fractions within a particular subcellular compartment, the total cellular amount of enzyme detectable by immunofluorescence was determined, and the results obtained for A and B were compared with each other. The data (Fig. 7) indicate that fractions A and B do not decay to a different extent. Injected dye-conjugated C-subunit fractions A and B decayed at a similar rate (not shown). Therefore, the N/C ratios indeed seem to reflect a different intracellular distribution.


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)

Decay of microinjected PKA C-subunit fraction A and B. NIH 3T3 cells were microinjected with purified PKA C-subunit fraction A or B at 4 mg/ml. At different time points after injection, cells were fixed and stained by immunofluorescence for injected PKA and subsequently mounted on glass slides. Images of injected cells were acquired as described in Materials and Methods. The average PKA-specific fluorescence intensity (including the nucleus and cytoplasm) in injected cells was determined, and the value obtained for cells fixed immediately after injection (t = 0) was set at 100%. The averages and SDs of two independent experiments with at least 65 cells measured are shown. Degradation of the injected PKA proteins appeared to be similar for the fraction A and B.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169370&req=5

Figure 7: Decay of microinjected PKA C-subunit fraction A and B. NIH 3T3 cells were microinjected with purified PKA C-subunit fraction A or B at 4 mg/ml. At different time points after injection, cells were fixed and stained by immunofluorescence for injected PKA and subsequently mounted on glass slides. Images of injected cells were acquired as described in Materials and Methods. The average PKA-specific fluorescence intensity (including the nucleus and cytoplasm) in injected cells was determined, and the value obtained for cells fixed immediately after injection (t = 0) was set at 100%. The averages and SDs of two independent experiments with at least 65 cells measured are shown. Degradation of the injected PKA proteins appeared to be similar for the fraction A and B.
Mentions: To evaluate the possibility that the apparent differences of the N/C ratios obtained with the fractions A and B were caused by a preferential degradation of one of the fractions within a particular subcellular compartment, the total cellular amount of enzyme detectable by immunofluorescence was determined, and the results obtained for A and B were compared with each other. The data (Fig. 7) indicate that fractions A and B do not decay to a different extent. Injected dye-conjugated C-subunit fractions A and B decayed at a similar rate (not shown). Therefore, the N/C ratios indeed seem to reflect a different intracellular distribution.

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