Paramecium Has Two Regulatory Subunits of Cyclic AMP‐Dependent Protein Kinase, One Unique to Cilia

Abstract

The subunit composition and intracellular location of the two forms of cAMP-dependent protein kinase of Paramecium cilia were determined using antibodies against the 40-kDa catalytic (C) and 44-kDa regulatory (R44) subunits of the 70-kDa cAMP-dependent protein kinase purified from deciliated cell bodies. Both C and R44 were present in soluble and particulate fractions of cilia and deciliated cells. Crude cilia and a soluble ciliary extract contained a 48-kDa protein (R48) weakly recognized by one of several monoclonal antibodies against R44, but not recognized by an anti-R44 polyclonal serum. Gel-filtration chromatography of a soluble ciliary extract resolved a 220-kDa form containing C and R48 and a 70-kDa form containing C and R44. In the large enzyme, R48 was the only protein to be autophosphorylated under conditions that allow autophosphorylation of R44. The subunits of the large enzyme subsequently were purified to homogeneity by cAMP-agarose chromatography. Both C and R48 were retained by the column and eluted with I M NaCl; no other proteins were purified in this step. These results confirm that the ciliary cAMP-dependent protein kinases have indistinguishable C subunits, but different R subunits. The small ciliary enzyme, like the cell-body enzyme, contains R44, whereas R48 is the R subunit of the large enzyme.

Cholangiocyte primary cilia are chemosensory organelles that detect biliary nucleotides via P2Y12 purinergic receptors

“…The localization of PKA and EPAC to cholangiocyte cilia may suggest the interconnectivity between EPAC-and PKA-mediated signaling in these organelles. PKA and AKAPs were previously found in Paramecium cilia (18), in mammalian motile cilia (25,44), and in the Chlamydomonas flagella (60,61). In this work, we demonstrate for the first time the expression of PKA, EPAC, and AKAP in mammalian primary cilia.…”

A novel, calcium‐inhibitable casein kinase in Paramecium cells

“…In some cases, ATP was replaced by 120 uM GTP including [y-32 P]GTP (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Reactions were started by adding protein kinase samples and, after 20 min at 20°C, terminated and measured by spotting 20 ul aliquots either onto trichloroacetic acid-prepared Whatman 1 Chr 3 MM filter papers [42] or, in the case of phosphate incorporation into peptide, by binding to phosphocellulose paper (Whatman P81) as described previously [44].…”

“…Standard assays for protein kinases were performed in a volume of 0.03 ml containing 5 mM MgCl 2 , 120 uM ATP, [y-32 P]ATP (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) MBq/umol), 19.8 ug mixed casein and buffer (20 mM triethanolamine-HCl buffer, 10% glycerol, 1 mM DTE, pH 7.5) with or without Ca 2+ as indicated. In some cases, ATP was replaced by 120 uM GTP including [y-32 P]GTP (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25).…”

“…In Paramecium tetraurelia several protein kinases have been isolated from cilia and cell bodies, such as cyclic nucleotidedependent [17][18][19][20] and Ca 2+ -dependent protein kinases [21,22]. Recently, three monomeric forms of CK-1 in Paramecium were reported [23], with molecular masses of 28^15 kDa, as well as an additional casein kinase of 65 kDa enriched in particulate fractions of whole cells and in cell cortices.…”

Adenosine activation of A2Breceptor(s) is essential for stimulated epithelial ciliary motility and clearance

“…Both of these receptors are members of the G protein-coupled receptor class and require activation of adenylyl cyclase that couples to the G-␣ subunit (2,3,42). Engagement of these receptors leads to changes in intracellular cAMP and calcium levels, both essential for increased CBF activity (9,24,39,44). Our laboratory previously demonstrated that cAMP-dependent phosphorylation via activation of PKA mediates increased CBF in ciliated BBECs (2,51,61,62).…”