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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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Comparison of the cellular distribution of microinjected PKA C-subunit fractions A and B conjugated with rhodamine or fluorescein. NIH 3T3 cells were microinjected at room temperature with labeled C-subunit fractions adjusted to 1 mg/ml. After a 10-min postinjection incubation at 37°C in the presence of 1 mM 8-bromo-cAMP, cells were fixed and the nuclear to cytoplasmic fluorescence was determined as described in Materials and Methods. The averages and SDs of two experiments with at least 60 cells quantified per group are shown.
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Figure 3: Comparison of the cellular distribution of microinjected PKA C-subunit fractions A and B conjugated with rhodamine or fluorescein. NIH 3T3 cells were microinjected at room temperature with labeled C-subunit fractions adjusted to 1 mg/ml. After a 10-min postinjection incubation at 37°C in the presence of 1 mM 8-bromo-cAMP, cells were fixed and the nuclear to cytoplasmic fluorescence was determined as described in Materials and Methods. The averages and SDs of two experiments with at least 60 cells quantified per group are shown.

Mentions: If the minor molecular differences between fraction A and B are responsible for a differential intracellular distribution, the type of dye conjugated might influence the distribution as well. Therefore, fraction A and B were conjugated with a different dye, rhodamine (TRITC), and microinjected into the cytoplasm of NIH 3T3 cells held subsequently in the presence of 8-bromo-cAMP. The N/C ratios obtained with the TRITC-conjugated fraction A and B 10 min after injection in comparison with the data obtained with FITC-conjugated enzyme fractions are shown in Fig. 3. TRITC-conjugated fraction B reaches an N/C ratio 2.3 times larger than TRITC-conjugated fraction A. The comparable relationships of the N/C ratios indicate that the enzyme distribution approached a similar balance independent of the type of dye conjugated.


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)

Comparison of the cellular distribution of microinjected PKA C-subunit fractions A and B conjugated with rhodamine or fluorescein. NIH 3T3 cells were microinjected at room temperature with labeled C-subunit fractions adjusted to 1 mg/ml. After a 10-min postinjection incubation at 37°C in the presence of 1 mM 8-bromo-cAMP, cells were fixed and the nuclear to cytoplasmic fluorescence was determined as described in Materials and Methods. The averages and SDs of two experiments with at least 60 cells quantified per group are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169370&req=5

Figure 3: Comparison of the cellular distribution of microinjected PKA C-subunit fractions A and B conjugated with rhodamine or fluorescein. NIH 3T3 cells were microinjected at room temperature with labeled C-subunit fractions adjusted to 1 mg/ml. After a 10-min postinjection incubation at 37°C in the presence of 1 mM 8-bromo-cAMP, cells were fixed and the nuclear to cytoplasmic fluorescence was determined as described in Materials and Methods. The averages and SDs of two experiments with at least 60 cells quantified per group are shown.
Mentions: If the minor molecular differences between fraction A and B are responsible for a differential intracellular distribution, the type of dye conjugated might influence the distribution as well. Therefore, fraction A and B were conjugated with a different dye, rhodamine (TRITC), and microinjected into the cytoplasm of NIH 3T3 cells held subsequently in the presence of 8-bromo-cAMP. The N/C ratios obtained with the TRITC-conjugated fraction A and B 10 min after injection in comparison with the data obtained with FITC-conjugated enzyme fractions are shown in Fig. 3. TRITC-conjugated fraction B reaches an N/C ratio 2.3 times larger than TRITC-conjugated fraction A. The comparable relationships of the N/C ratios indicate that the enzyme distribution approached a similar balance independent of the type of dye conjugated.

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