The biological significance of the NH2
-terminal domain of the PKA C-subunit, a segment comprising ~40 residues that precedes the catalytic domain, is unknown. In the crystal structure, it forms an amphipathic α-helix (Veron et al. 1993
) and reaches from the large to the small lobe of the enzyme opposite the catalytic cleft (Knighton et al. 1991
; Bossemeyer et al. 1993
). The NH2
Gly-Asn-Ala is conserved among the isozymes Cα, Cβ, and Cγ, the latter being expressed in the testis (Beebe et al. 1990
). We have shown previously that Cα and Cβ are prone to deamidation at Asn2, since about one-third of the C-subunit isolated from striated muscle of four different mammalian species (pig, cattle, rabbit, and rat) are in the myr
Gly-Asp-Ala configuration, which is an NH2
-terminal sequence not so far present in protein data banks. There is evidence that deamidation of the C-subunit occurs in vivo (Jedrzejewski et al. 1998
). The physiological significance of the Asp2 form is certainly not limited to the fact that it is more resistant towards a specific protease isolated from kidney brush border membranes than the Asn2 form (Kinzel et al. 1987
). The finding that after microinjection, the Asn2 form reaches a significantly larger nuclear/cytoplasmic ratio than the Asp2 form constitutes evidence that the NH2
terminus of the C-subunit is critical for the intracellular distribution of the enzyme. This difference between the encoded and the deamidated form has been observed regardless of whether the C-subunit was labeled with FITC, TRITC, or was in unlabeled form. The N/C ratio in the plateau phase with the Asn2 form was ~1.5–2.3 times greater than that observed with the Asp2 form. The difference in the intracellular distribution appears to be independent of the mammalian species, as enzymes from two different sources behave in the same manner, and the molecular parameters from two further mammalian species reproduce those seen with the former (Jedrzejewski et al. 1998
). This apparent independence of the data from the cell type used suggests a general mechanism designed to discriminate between the two forms of the enzyme.
The relatively large amounts of microinjected C-subunit fractions required to measure the intracellular distribution may question the potential physiological significance of the results. However, the significance is supported by data on the site-specific phosphorylation of nuclear CREB, a reaction known to activate the transcription factor, elicited by the small enzyme amounts injected. The differential phosphorylation by fraction A and B is particularly well demonstrable at the small enzyme amounts, which yield intracellular concentrations in the physiological range. Under these conditions, nuclear substrate is probably not a limiting factor. The encoded, Asn 2 form of the PKA C-subunit is more efficient in that respect than the deamidated, Asp2 form. Since in vitro both enzyme fractions were shown to phosphorylate PKA substrates (Kinzel et al. 1987
), including CREB equally effective, the data indicate that the differences observed in vivo are based on the uneven intracellular distribution; i.e., the larger nuclear level of the encoded C-subunit. The distribution data and the degree of protein phosphorylation in the nucleus correlate. Therefore, the differential distribution of both enzyme fractions observed at high concentrations is unlikely to be caused by this fact, but appears to reflect a physiological relevance.
The difference in the N/C ratios observed for fraction A and B might suggest in the first instance that it is the increase in electronegativity that causes a decrease of the nuclear enzyme level of fraction A. A simple charge difference as the driving force for the variant distribution of the Asn2 and the Asp2 form is unlikely, however, because of the fact that FITC labeling renders fraction B even more acidic than the unlabeled fraction A (), yet it, nevertheless, reaches larger N/C values than the latter. Therefore, it seems that a negative charge alone is not sufficient to explain the decrease in the nuclear level of the enzyme, but that it must probably be added at a specific site.
The Asn2 residue per se does not appear to constitute a determinant for the intracellular enzyme distribution as its deletion does not alter the N/C ratio of the deletion mutant when compared with the wild-type enzyme. It appears that Asn2 plays a role as a deamidation site only, conserved in Cα, Cβ, and Cγ and, thus, as a potential source for increasing electronegativity at the NH2
terminus, and perhaps with the further consequence of facilitating Ser10 autophosphorylation. Since a negatively charged residue next to Gly1 inhibits N
-myristoylation (Towler et al. 1988
), deamidation appears to be the only way to achieve the specific sequence myr
Gly1-Asp2 observed in fraction A.
The gain in electronegativity close to the NH2
-terminal myristoyl moiety in proteins is reminiscent of myristoyl-electrostatic switch proteins (for review see, McLaughlin and Aderem 1995
) in which electronegativity is achieved through phosphorylation aimed at influencing the interaction of these proteins with membranes. This is usually mediated by the myristoyl moiety partitioning into the lipid bilayer with positively charged residues interacting with acidic phospholipid head groups. Thus, it is possible that a lipophilic entity of membranous or proteinaceous nature may participate in the recognition and in the determination of the intracellular distribution of the C-subunit. It has been shown that the NH2
terminus of the PKA C-subunit in chimeric pp60v-src
can partially substitute for the wild-type NH2
terminus in conferring membrane binding in vivo (Silverman and Resh 1992
). However, this observation with the myristoylated NH2
terminus of the C-subunit within the foreign structural context of another protein may not necessarily reflect its natural role in PKA. A PKA-specific significance of its myristoylated NH2
terminus may be indicated by the finding that the rate of C-subunit binding to liposomes in vitro is dramatically increased on interaction with the regulatory subunit type II (RII) but not with type I (Gangal et al. 1999
). Even these PKA-specific observations, however, are unlikely to play a role since the experiments in our study were carried out under saturating amounts of cAMP and with excess of free C-subunit sufficient to guarantee subunit dissociation and to exclude a role of R-subunit isoforms.
Results published so far on the cytoplasmic to nuclear translocation of the free C-subunit are compatible with a model in which the enzyme can traverse nuclear pores in either direction by diffusion (Harootunian et al. 1993
). A functional nuclear localization signal has not been identified yet in C-subunits. In agreement with a diffusion model, the distribution is not particularly influenced by the concentration of the C-subunit. In our study, the unlabeled enzymes were injected at a four times larger concentration than the labeled enzymes. The N/C ratios of the plateau, however, reach comparable values characteristic for a specific isoform, indicating that it is not saturable, at least not within this range. However, the observation that both forms of the enzyme after ~10 min reach an equilibrium which is different for the deamidated and the nondeamidated forms, seems to require supplementary assumptions for explanation.
Deamidation of Asn2 might, of course, result in changes of the higher ordered structure of the NH2
terminus. No noticeable structural differences are apparent between the high resolution structure of the fraction A enzyme (Bossemeyer et al. 1993
) and the recombinant bovine (Engh et al. 1996
) or mouse (Knighton et al. 1991
) enzyme; however, the immediate area around Asn2 is not well resolved in the ternary structure (Bossemeyer et al. 1993
; Zheng et al. 1993
). Comparative secondary structure studies of synthetic myristoylated hexadecapeptides representing the bovine Cα NH2
terminus either in the Asn2 and the Asp2 form provided no evidence for significant structural differences at this level (Tholey, A., D. Bossemeyer, V. Kinzel, and J. Reed, unpublished data). This still does not mean that a different structure or flexibility might not occur. It could be induced by interaction with cellular target(s), a possibility indicated indirectly by RII-induced binding of C-subunits to liposomes in vitro, which was not seen with RI (Gangal et al. 1999
). The charge shift to a more acidic character of the NH2
terminus could be sufficient to result in a preference for cytoplasmic structures that would retain fraction A to a certain extent in the cytoplasm, or a more favorable environment for the less acidic fraction B in the nucleus. The described differences in the sensitivity of fraction A and B to a specific protease in vitro (Kinzel et al. 1987
) suggests that such differences are in principle recognizable at the molecular level. A differential export from the nucleus (Schmidt-Zachmann et al. 1993
), mediated in the case of the C-subunit by protein kinase inhibitor protein (PKI) (Fantozzi et al. 1994
), represents another possibility that requires further attention.
The fact that the different N/C ratios for fraction A and B are seen in the presence of saturating amounts of cAMP seems to argue against R-subunits as a retaining principle. The interaction of enzyme fractions A and B with RI or RII appeared to be indistinguishable in vitro (Kinzel et al. 1987
). However, it is not excluded that the affinity of type II R-subunits for C-subunits in situ may be modulated by association with anchor proteins (Scott 1991
; Hubbard and Cohen 1993
) in such a way that the isozymes are bound to a different extent. A preferential binding of cell-endogenous PKI to the Asp2 form of the enzyme seems unlikely, however, as the cause for differential nuclear uptake since both enzyme fractions appeared to interact equally well with PKI, at least in vitro (Kinzel et al. 1987
; Van Patten et al. 1988
). Again, the isozymes might bind to cytoplasmic substrates to a different extent. The finding that the phosphorylation by fraction A and B of standard substrates including CREB in vitro was identical does not reflect the situation in the living cell (Lamb et al. 1988
, Lamb et al. 1989
; this study).
The example of the C-subunit may be typical of cases where proteins fulfill nuclear as well as cytoplasmic functions. Such proteins may adapt intermediate states of intracellular distribution by different mechanisms. One of them seems to be site-specific deamidation, as shown in this study. The circumstances in cells that control the partial deamidation of certain proteins are unknown and may even differ among candidate proteins; those for the C-subunit are unknown as well. However, they may play an important role in those cells or tissues in which the C-subunit is required to fulfill specific cytoplasmic functions. A preferentially deamidated state of the C-subunit may explain the lack of a significant redistribution of the enzyme to the nucleus upon treatment of certain cells with 8-bromo-cAMP (Murtaugh et al. 1982
). The occurrence of comparable fraction A/fraction B ratios in muscle tissues of different mammalian species (Kinzel et al. 1987
; Jedrzejewski et al. 1998
) seems to point to a physiological significance of this modification. An example of protein-mediated deamidation of a cellular protein at a specific glutamine residue caused by a bacterial toxin has been described recently (Flatau et al. 1997
; Schmidt et al. 1997
). Moreover, several myristoylated proteins involved in signal transduction share the NH2
-Gly-Asn sequence and, thus, may be candidates for partial deamidation (listed by Jedrzejewski et al. 1998
Although the iso
Asp2 form of the enzyme cannot be evaluated separately, its presence in trace amounts in fraction A is worth mentioning in the context of this study. The occurrence of iso
Asp2 in the C-subunit fraction A points to a mechanism of deamidation through the so-called β-aspartyl-shift mechanism (for review see Wright 1991
). It involves the formation of a cyclic succinimide intermediate that opens to yield preferentially the iso
Asp form (to a lesser extent the Asp form) (for reviews see Bornstein and Balian 1977
; Aswad and Johnson 1987
; Geiger and Clarke 1987
; Lura and Schirch 1988
; Artigues et al. 1990
), which carries the original Cβ-methylene of Asn2 now in the peptide backbone. The inverse ratio of iso
Asp/Asp in tissues, with a clear-cut surplus of the Asp form, is probably due to the action of a ubiquitous repair enzyme, protein carboxyl methyltransferase, which methylates the atypical α-carboxyl group of l
Asp residues (Aswad 1984
; Murray and Clarke, 1989) as a part of a mechanism aimed at the reconstitution of the regular peptide backbone. For a more detailed discussion of this relation with respect to the C-subunit see Jedrzejewski et al. 1998
. Cases of site-specific nonenzymatic deamidation of proteins have been reported, which appear to be involved in aging and subsequent degradation of proteins. However, in certain proteins, this type of modification appears to be part of their physiological program (for review see Wright 1991
). The conserved Asn2 deamidation of the PKA C-subunit with signs of ample repair (to yield predominantly the Asp 2 form) may belong to the latter group. This appears to be supported by the differential in vivo phosphorylation of the CREB family transcription factor(s) effected by the encoded and the deamidated C-subunit.
In conclusion, this study with living cells allows for the first time to consider different physiological functions of the major C-subunit isozymes of PKA (generated by deamidation of Asn2) on the basis of their differential distribution within cells, and points to a potential physiological significance of the NH2-terminal domain of this enzyme, and possibly of other proteins with a similar NH2 terminus for which the C-subunit may represent a model system. Further studies are aimed at the elucidation of the control of deamidation and of the molecular mechanism responsible for the altered intracellular distribution of Asn2 deamidated catalytic subunit.