Cyclic nucleotide-independent phosphorylation of Vitellin by casein kinase II purified from Rhodnius prolixus oocytes
Introduction
Vitellogenins (VGs) are high molecular weight phosphorylated lipoglycoproteins sequestered from insect hemolymph through receptor-mediated endocytosis and stored in growing oocytes during oogenesis (Sappington and Raikhel, 1998). These proteins will supply the nutrients required by the growing embryos following oocyte fertilization. Phosphorylation is one of the most recurrent covalent protein modifications known to occur to VG molecules during their synthesis. It has been described in every oviparous organism studied so far (Gottlieb and Wallace, 1981, Wang and Williams, 1982, Takahashi, 1987, Byrne, Gruber and Ab, 1989, Dhadialla and Raikhel, 1990). Extensive phosphorylation is particularly found in non-mammalian vertebrate VGs within a domain named phosvitin (Byrne et al., 1989). Once inside the oocytes VGs are cleaved due to the action of proteases and phosvitin then becomes an independent protein (Bergink and Wallace, 1974, Byrne, Gruber and Ab, 1989). Nearly half of its aminoacid composition is made up of serines and most of them are phosphorylated (Byrne, Gruber and Ab, 1989, Grogan, Shirazi and Taborsky, 1990). The protein kinase that phosphorylates phosvitin was first purified from the supernatant of rooster liver. It was then verified that a remarkable feature of the so-called phosvitin kinases was their insensitivity to cyclic nucleotides (Goldstein and Hasty, 1973, Dastugue, Tichonicky and Kruh, 1974).
Arthropod VGs are usually synthesized in extra-ovarian tissues such as the fat body (Valle, 1993, Sappington and Raikhel, 1998). Exceptions to this general rule include some species of Diptera and the Hemipteran Rhodnius prolixus where additional VG synthesis occurs in the ovaries (Valle, 1993, Melo, Valle, Machado, Salerno, Paiva-Silva, Cunha E Silva, Souza and Masuda, 2000). After extensive post-translational processing fat body-derived VGs are secreted into the hemolymph, sequestered by specific receptors on the ovarian surface and stored in intracellular deposits called yolk platelets (Byrne, Gruber and Ab, 1989, Dhadialla and Raikhel, 1990, Telfer et al, 1982, Raikhel and Dhadialla, 1992). VG inside the oocytes is conventionally named Vitellin (VT). Invertebrates VGs differ from their non-mammalian vertebrate counterparts in at least three aspects: first, they are phosphorylated to a lower degree, with a consequently smaller phosvitin-like domain (Romans, Tu, Ke and Hagedorn, 1995, Yano et al., 1994a, Trewitt, Heilmann, Degrugillier and Krishna Kumaran, 1992); second, they do not suffer extensive proteolytic processing after endocytosis; and third, post endocytotic phosphorylation may occur in some insect groups (Takahashi, 1984, Byrne, Gruber and Ab, 1989, Wang and Telfer, 2000).
VT phosphorylation after endocytosis in insects was first described in Bombyx mori (Takahashi, 1984, Takahashi, 1985, Takahashi, 1987). Enzymes involved in this process were isolated and characterized as two cyclic nucleotide-dependent protein kinases, protein kinase A (PK A) and protein kinase G (PK G). Activation of these enzymes relies on the increase of intracellular concentrations of c-AMP and c-GMP (Burgoyne and Petersen, 1997). Cyclic nucleotide production usually follows from either hormone binding to G protein coupled-receptors which then activate adenylate or guanylate cyclases or eventually from the activation of soluble guanylate cyclases by nitric oxide (Eppig, 1989, Denninger and Marletta, 1999). Wang and Telfer (1996) established a link between cyclic nucleotide concentration and the control of VG endocytosis in isolated ovarian follicles of Hyalophora cecropia. They have shown recently that the phosphorylation of a set of particle-associated proteins 27, 32 and 45 kDa strongly relies on cyclic nucleotide levels (Wang and Telfer, 2000). However, direct phosphorylation of VT subunits in that system seems to be catalyzed by a protein kinase independent of cyclic nucleotides (Wang and Telfer, 2000).
Casein kinase II (CK II) is a cyclic nucleotide-independent Ca2+/calmodulin-insensitive protein kinase that is inhibited in vitro by heparin and utilizes both ATP and GTP as phosphate donors (Pinna and Meggio, 1997). The term “casein kinase” has been operationally adopted to indicate a Ser/Thr protein kinase able to phosphorylate acidic substrates such as casein and phosvitin (Pinna and Meggio, 1997, Sarno, Marin, Boschetti, Pagano, Meggio and Pinna, 2000). This enzyme is composed of two catalytic α subunits and two regulatory β subunits, which form a tetrameric complex α2β2. CK II has a broad sprectrum distribution in organisms and is involved in countless intracellular processes which include the control of intermediary metabolism, cytoskeletal assembly, gene expression and cell cycle regulation (Pinna, 1990, Pinna and Meggio, 1997). However the control of its enzymatic activity by cellular components is far from understood. CK II recognizes phosphoaceptor sites that are specified by clusters of acidic residues such as glutamic and aspartic acid or phosphoserines as is the case of casein and phosvitin (Pinna and Meggio, 1997, Hrubey and Roach, 1990).
Silva-Neto and Oliveira (1993) demonstrated that chorionated oocyte homogenates of R. prolixus contain a protein kinase activity with the general properties of CK II. Fialho et al. (1999) showed that this maternally derived CK II activity is increased up to three-fold after oocyte fertilization and further development. In the present study we provide physical-chemical, immunological and biochemical evidence that CK II is the cyclic nucleotide-independent protein kinase able to phosphorylate oocyte VT in vitro. These results suggest that once inside the oocytes VT phosphorylation status can be altered by a housekeeping protein kinase activity. The possibility of CK II action as a regulator of VT properties during egg development is discussed.
Section snippets
Chemicals
SBTI, LBTI, leupeptin, benzamidine, antipain, PMSF, EDTA, EGTA, sodium azide, bovine serum albumin, heparin, ATP, c-AMP, molecular weight standards, Cellulose phosphate, pre-packed Heparin-Agarose, sphingosine and polylysine were from Sigma Chemical Company (St. Louis, MO). H-8, H-89 and calphostin C were purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA, USA). Polyclonal antibody against the α subunit of human CK II was from Upstate Biotechnology Incorporated (Lake Placid, NY, USA).
Inhibition of Vitellin phosphorylation in total oocyte supernatants
Cyclic nucleotide-dependent protein kinases such as PK A and PK G have been previously described both in the ovaries of Bombyx mori and Hyalophora cecropia (Takahashi, 1984, Takahashi, 1985, Takahashi, 1987, Wang and Telfer, 1996, Wang and Telfer, 2000). Rhodnius chorionated oocytes and developing eggs contain a protein kinase (CK II) whose activity is not sensitive to cyclic nucleotides (Silva-Neto and Oliveira, 1993, Fialho, Masuda and SilvaNeto, 1999). We decided then to verify which protein
Discussion
In this study we suggest that CK II is the main VT kinase in total chorionated oocyte supernatants of Rhodnius prolixus. The following evidence supports this statement. VT phosphorylation in vitro is totally abolished in the presence of heparin (Fig. 1). The main protein kinase present in the oocyte supernatant is retained in affinity chromatography on Heparin-Agarose (Fig. 2) and is recognized by antibodies raised against the human CK II (Fig. 2, inset). A CK II enriched preparation obtained
Acknowledgements
We thank Lilian S. C. Gomes, Rosane O. M. M. Costa, Heloísa S. L. Coelho, José S. Lima Junior and Litiane M. Rodrigues for excellent technical assistance. This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Universitária José Bonifácio (FUJB-ALV), Programa de Apoio ao Desenvolvimento Científico e Tecnológico (PADCT III), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). Dr. M.A.C. Silva-Neto
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