Differential distribution of cAMP-dependent protein kinase isoforms in the mantle of the bivalve mollusc Mytilus galloprovincialis.

Two different isoforms of the regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), had previously been identified in the sea mussel Mytilus galloprovincialis. R(myt1) and R(myt2) were differentially distributed in the various cell types comprising the mussel mantle. R(myt1) was found the only isoform to be present in the auxiliary cells of female follicles and the cubic epithelium of the middle fold of mantle edge. In contrast, only the R(myt2) isoform was detected in the subepithelial connective tissue, the endothelium of haemolymph vessels, the adipogranular and vesicular cells of reserve connective tissue, and the spermatozoa. Finally, both R(myt1) and R(myt2) coexist in some cell types but they show a different cellular localization: R(myt1) was localized in the cytoplasm whereas R(myt2) was mainly detected in the cell apical edge, cell periphery, cilia and sperm flagella. Interestingly, both R(myt1) and R(myt2) were absent in oocytes within the female gonadal follicles but, in contrast, they were highly expressed in follicle-released oocytes collected after spawning. Taken together the results show that mussel PKA isoforms are differentially distributed in the mantle cell types, which suggests that they are involved in the regulation of distinct cellular functions. On the other hand, the expression of R(myt1) and R(myt2) proteins is associated with the meiosis resumption of oocytes at the prophase I stage, which occurs in parallel to spawning.

Isoforms of cAMP-dependent protein kinase in the bivalve mollusk Mytilus galloprovincialis: activation by cyclic nucleotides and effect of temperature.

Two different isoforms of cAMP-dependent protein kinase (PKA) have been partially purified from the posterior adductor muscle and the mantle tissue of the sea mussel Mytilus galloprovincialis. The holoenzymes contain as regulatory subunit (R) the previously identified isoforms Rmyt1 and Rmyt2, and were named PKAmyt1 and PKAmyt2, respectively. Both cAMP and cGMP can activate these PKA isoforms completely, although they exhibit a sensitivity approximately 100-fold higher for cAMP than for cGMP. When compared to PKAmyt2, the affinity of PKAmyt1 for cAMP and cGMP is 2- and 3.5-fold higher, respectively. The effect of temperature on the protein kinase activity of both PKA isoforms was examined. Temperature changes did not affect significantly the apparent activation constants (Ka) for cAMP. However, the protein kinase activity was clearly modified and a remarkable difference was observed between both PKA isoforms. PKAmyt1 showed a linear Arrhenius plot over the full range of temperature tested, with an activation energy of 15.3+/-1.5 kJ/mol. By contrast, PKAmyt2 showed a distinct break in the Arrhenius plot at 15 degrees C; the activation energy when temperature was above 15 degrees C was 7-fold higher than that of lower temperatures (70.9+/-8.1 kJ/mol vs 10.6+/-6.5 kJ/mol). These data indicate that, above 15 degrees C, PKAmyt2 activity is much more temperature-dependent than that of PKAmyt1. This different behavior would be related to the different role that these isoforms may play in the tissues where they are located.

Characterization of a type I regulatory subunit of cAMP-dependent protein kinase from the bivalve mollusk Mytilus galloprovincialis.

Two isoforms of the regulatory subunit (R) of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), had been purified in our laboratory from two different tissues of the sea mussel Mytilus galloprovincialis. In this paper, we report the sequences of several peptides obtained from tryptic digestion of R(myt1). As a whole, these sequences showed high homology with regions of type I R subunits from invertebrate and also from mammalian sources, but homology with those of fungal and type II R subunits was much lower, which indicates that R(myt1) can be considered as a type I R isoform. This conclusion is also supported by the following biochemical properties: (1) R(myt1) was proved to have interchain disulfide bonds stabilizing its dimeric structure; (2) it failed to be phosphorylated by the catalytic (C) subunit purified from mussel; (3) it has a higher pI value than that of the R(myt2) isoform; and (4) it showed cross-reactivity with mammalian anti-RIbeta antibody.