Multigene Family Encoding 3′,5′-Cyclic-GMP-Dependent Protein Kinases in Paramecium tetraurelia Cells

Abstract

In the ciliate Paramecium tetraurelia, 3,5-cyclic GMP (cGMP) is one of the second messengers involved in several signal transduction pathways. The enzymes for its production and degradation are well established for these cells, whereas less is known about the potential effector proteins. On the basis of a current Paramecium genome project, we have identified a multigene family with at least 35 members, all of which encode cGMPdependent protein kinases (PKGs). They can be classified into 16 subfamilies with several members each. Two of the genes, PKG1-1 and PKG2-1, were analyzed in more detail after molecular cloning. They encode monomeric enzymes of 770 and 819 amino acids, respectively, whose overall domain organization resembles that in higher eukaryotes. The enzymes contain a regulatory domain of two tandem cyclic nucleotide-binding sites flanked by an amino-terminal region for intracellular localization and a catalytic domain with highly conserved regions for ATP binding and catalysis. However, some Paramecium PKGs show a different structure. In Western blots, PKGs are detected both as cytosolic and as structure-bound forms. Immunofluorescence labeling shows enrichment in the cell cortex, notably around the dense-core secretory vesicles (trichocysts), as well as in cilia. Immunogold electron microscopy analysis reveals consistent labeling of ciliary membranes, of the membrane complex composed of cell membrane and cortical Ca 2؉ stores, and of regions adjacent to ciliary basal bodies, trichocysts, and trafficking vesicles. Since PKGs (re)phosphorylate the exocytosis-sensitive phosphoprotein pp63/pf upon stimulation, the role of PKGs during stimulated exocytosis is discussed, in addition to a role in ciliary beat regulation.In eukaryotic cells, 3Ј,5Ј-cyclic GMP (cGMP) is one of the second messengers participating in signal transduction pathways. Its significance has been only partially elucidated. Three effector activities are currently discussed (48, 77), namely, (i) cGMP-induced activation of ion channels, (ii) cGMP-sensitive phosphodiesterase (PD) activity, and (iii) Ser/Thr-specific cGMP-dependent protein kinase (PKG) activity. PKGs are found not only in metazoans of different phyla (48, 60) but also in flowering plants (73) and lower eukaryotes (16,23). The metazoan PKGs can be divided into two types, a soluble type I form with several splice forms (58,66,83) and a single membrane-bound type II form (33,76). Unlike mammalian and invertebrate PKGs, which are typically homodimers, the protozoan PKGs known so far are all monomeric proteins (16,23,55,80). The multiple interactions of PKGs with other signaling systems (77) are particularly intriguing. Signaling via PKGs presupposes the occurrence of a guanylate cyclase (GC) and termination by PD activity.Among potential signaling components, the following are known to occur in the ciliate Paramecium: a Ca 2ϩ -dependent GC (47, 67), PD (68), and a PKG (53). The broad spectrum of cellular activities governed or modulated by PKGs led us to identify the correspond...

Cross-study analysis of genomic data defines the ciliate multigenic epiplasmin family: strategies for functional analysis in Paramecium tetraurelia

“…Within this multigenic family, maintenance of genes during the successive genome duplications is in good agreement with expected behavior of genes subject to dosage constraints (highly expressed genes are preferentially retained as duplicates[ 9 ]). Most of the sub-groups observed in the epiplasmin family have a 4+2 topology, a distribution also described for other sets of highly expressed proteins: 35 cGMP-dependent protein kinases [ 19 ], 26 syntaxins [ 20 ], 12 synaptobrevins [ 21 ] and 17 vacuolar-proton-ATPases [ 22 ].…”

Regulation of the Na+‐K+‐2Cl− cotransporter by cGMP/cGMP‐dependent protein kinase I after furosemide administration

“…The involvement of cGMP/PKG in membrane trafficking via interactions with SNARE proteins is also discussed by Kissmehl et al [21] and Yokoyama et al [22] but remains unexplored in TAL cells.…”

Molecular aspects of rapid, reversible, Ca2+-dependent de-phosphorylation of pp63/parafusin during stimulated exo-endocytosis in Paramecium cells

“…In the Paramecium cell cortex, any Ca 2 +-release channels would have to be located on the outer part of alveolar sacs, because the part facing the cell center is densely studded with Ca 2 +-pump molecules [56]. In agreement with this postulate, the subplasmalemmal space, including that delineated by alveolar sacs and trichocysts, is the site where pp63/pf [36], as well as the relevant protein phosphatase PP2B/CaN [42] and protein kinases CK-2 and PKG [57,58] are heavily enriched according to immuno-EM studies (see Fig. 1 and below).…”

“…Cloning and immunogold EM localisation revealed that guanylate cyclase is associated with the (ciliary and non-ciliary) cell membrane and the alveolar sacs membrane complex [63]. Interestingly this is also the distribution of the cGMP-activated protein kinase, PKG [58] (Fig. 1).…”