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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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Isoelectric focusing results obtained with native porcine PKA C-subunit, before (lane 1) and after separation in fraction A (lane 2) and fraction B (lane 3). Fluorescein-labeled fractions A and B are shown in lane 4 and 5 respectively. Arrows indicate the native and the predominantly labeled fraction A (▸) and B (▹).
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Figure 4: Isoelectric focusing results obtained with native porcine PKA C-subunit, before (lane 1) and after separation in fraction A (lane 2) and fraction B (lane 3). Fluorescein-labeled fractions A and B are shown in lane 4 and 5 respectively. Arrows indicate the native and the predominantly labeled fraction A (▸) and B (▹).

Mentions: The possibility that the charge differences between both enzyme fractions could contribute to the differential translocation behavior led to a control of the isoelectric values of conjugated enzymes. Labeling with rhodamine as well as fluorescein lowered the isoelectric point of the respective enzymes by >0.4 pH units. The data obtained with FITC-conjugated C-isozymes are illustrated in Fig. 4 (lanes 4 and 5). Lane 1 shows the unlabeled electrophoretically homogenous C-subunit preparation composed of the two charge variants. Isolated native fraction A (lane 2) and fraction B (lane 3) focus at about pH 7.1 and 7.5, respectively. (The isoelectric values of Cα and Cβ isozymes were indistinguishable.) Occasionally, fraction B may contain traces of the more acidic A fraction because of some spillover on the column. Upon FITC conjugation of fraction A (lane 4) and fraction B (lane 5) (labeling ratio 0.9, respectively, 1.3 mol FITC/mol C-subunit), the major amount of protein is shifted in both cases towards a more acidic value. In the case of fraction B (lane 5), it should be noted that the major portion of the labeled enzyme is more acidic than the unlabeled fraction A, and that a further protein band appears at an even lower pH, possibly representing enzyme with 2 mol FITC bound per mole.


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)

Isoelectric focusing results obtained with native porcine PKA C-subunit, before (lane 1) and after separation in fraction A (lane 2) and fraction B (lane 3). Fluorescein-labeled fractions A and B are shown in lane 4 and 5 respectively. Arrows indicate the native and the predominantly labeled fraction A (▸) and B (▹).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Isoelectric focusing results obtained with native porcine PKA C-subunit, before (lane 1) and after separation in fraction A (lane 2) and fraction B (lane 3). Fluorescein-labeled fractions A and B are shown in lane 4 and 5 respectively. Arrows indicate the native and the predominantly labeled fraction A (▸) and B (▹).
Mentions: The possibility that the charge differences between both enzyme fractions could contribute to the differential translocation behavior led to a control of the isoelectric values of conjugated enzymes. Labeling with rhodamine as well as fluorescein lowered the isoelectric point of the respective enzymes by >0.4 pH units. The data obtained with FITC-conjugated C-isozymes are illustrated in Fig. 4 (lanes 4 and 5). Lane 1 shows the unlabeled electrophoretically homogenous C-subunit preparation composed of the two charge variants. Isolated native fraction A (lane 2) and fraction B (lane 3) focus at about pH 7.1 and 7.5, respectively. (The isoelectric values of Cα and Cβ isozymes were indistinguishable.) Occasionally, fraction B may contain traces of the more acidic A fraction because of some spillover on the column. Upon FITC conjugation of fraction A (lane 4) and fraction B (lane 5) (labeling ratio 0.9, respectively, 1.3 mol FITC/mol C-subunit), the major amount of protein is shifted in both cases towards a more acidic value. In the case of fraction B (lane 5), it should be noted that the major portion of the labeled enzyme is more acidic than the unlabeled fraction A, and that a further protein band appears at an even lower pH, possibly representing enzyme with 2 mol FITC bound per mole.

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