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

The cAMP signalling pathway is involved in the regulation of basic physiological processes in bivalve molluscs. We had previously identified and characterized two isoforms of cAMP-dependent protein kinase (PKA) from the sea mussel Mytilus galloprovincialis that differ at their regulatory (R) subunit, namely, R(myt1) or R(myt2). Here we investigated the immunohistochemical expression of both PKA isoforms in various mussel tissues. R(myt1) and R(myt2) displayed a complementary subcellular localization. In general, R(myt1) was found to be uniformly distributed in the cytoplasm of most cell types, whereas R(myt2) appears to be localized only in the cell periphery and associated with certain cellular structures, such as the cilia of labial palps and gill filaments. Thus, both PKA isoforms appear to be non-redundant, but they have specific functions. R(myt1) was the main isoform present in catch muscle fibers, which suggests that PKA(myt1) may be the isoform involved in the regulation of the catch state. Conversely, R(myt2) was the only isoform detected in the cilia of gill filaments, indicating that PKA(myt2) could mediate the effects of cAMP on the ciliary beat frequency.

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.

Identification of multiple isoforms of the cAMP-dependent protein kinase catalytic subunit in the bivalve mollusc Mytilus galloprovincialis.

Several isoforms of the cAMP-dependent protein kinase catalytic subunit (C-subunit) were separated from the posterior adductor muscle and the mantle tissues of the sea mussel Mytilus galloprovincialis by cation exchange chromatography, and identified by: (a) protein kinase activity; (b) antibody recognition; and (c) peptide mass fingerprinting. Some of the isozymes seemed to be tissue-specific, and all them were phosphorylated at serine and threonine residues and showed slight but significant differences in their apparent molecular mass values, which ranged from 41.3 to 44.5 kDa. The results from the MS analysis suggest that at least some of the mussel C-subunit isoforms arise as a result of alternative splicing events. Furthermore, several peptide sequences from mussel C-subunits, determined by de novo sequencing, showed a high degree of homology with the mammalian Calpha-isoform, and contained some structural motifs that are essential for catalytic function. On the other hand, no significant differences were observed in the kinetic parameters of C-subunit isoforms, determined by using synthetic peptides as substrate and inhibitor. However, the C-subunit isoforms separated from the mantle tissue differed in their ability to phosphorylate in vitro some proteins present in a mantle extract.

CK2-mediated phosphorylation of a type II regulatory subunit of cAMP-dependent protein kinase from the mollusk Mytilus galloprovincialis.

Two isoforms of regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), were identified so far in the sea mussel Mytilus galloprovincialis. Out of them, only R(myt2) was phosphorylated in vitro by casein kinase 2 (CK2) using GTP as phosphate donor. CK2 catalytic subunit (CK2alpha) itself was sufficient to phosphorylate R(myt2), but phosphorylation was enhanced by the presence of the regulatory subunit CK2beta. Even in the absence of CK2, R(myt2) was phosphorylated to a certain extent when it was incubated with GTP. This basal phosphorylation was partially abolished by the known inhibitors apigenin and emodin, which suggests the presence of a residual amount of endogenous CK2 in the preparation of purified R subunit. CK2-mediated phosphorylation significantly decreases the ability of R(myt2) to inhibit PKA catalytic (C) subunit activity in the absence of cAMP. On the other hand, the sequence of several peptides obtained from the tryptic digestion of R(myt2) showed that mussel protein contains the signature sequence common to all PKA family members, within the "phosphate binding cassette" (PBC) A and B. Moreover, the degree of identity between the sequences of peptides from R(myt2), as a whole, and those from type II R subunits was 68-75%, but the global identity percentage with type I R subunits was only about 30%, so that R(myt2) can be classified as a type II R subunit.

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.