R. Pepperkok et al., Intracellular distribution of mammalian protein kinase A catalytic subunitaltered by conserved Asn2 deamidation, J CELL BIOL, 148(4), 2000, pp. 715-726
The catalytic (C) subunit of protein kinase A functions both in the cytopla
sm 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 NH2-terminal sequence myrGly-Asn-Ala (Jedrzejewski
, P.T., A. Girod, A. Tholey, N. Konig, S. Thullner, V. Kinzel, and D. Bosse
meyer. 1998. Protein Sci. 7:457-469). Because of the increase of electroneg
ativity by generation of Asp2, it is reminiscent of a myristoyl-electrostat
ic switch. To compare the intracellular distribution of the enzymes, both f
orms of porcine or bovine heart enzyme were microinjected into the cytoplas
m of mouse NIH 3T3 cells after conjugation with fluorescein, rhodamine, or
in unlabeled form, The nuclear/cytoplasmic fluorescence ratio (N/C) was ana
lyzed in the presence of cAMP (in the case of unlabeled enzyme by antibodie
s). Under all circumstances, the NIC ratio obtained with the encoded Asn2 f
orm 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 intr
acellular distribution of both enzyme forms is also reflected by functional
data. It correlates with the degree of phosphorylation of the key serine i
n CREB family transcription factors in the nucleus. Microinjection of myris
toylated recombinant bovine C alpha and the Asn2 deletion mutant of it yiel
ded N/C ratios in the same range as encoded native enzymes, Thus, Asn2 seem
s to serve as a potential site for modulating electronegativity, The data i
ndicate that the NH2-terminal domain of the PKA C-subunit contributes to th
e intracellular distribution of free enzyme, which can be altered by site-s
pecific in vivo deamidation, The model character for other signaling protei
ns starting with myrGly-Asn is discussed.