Purification and Phosphorylation of a Mr 25, 000 Protein, an Effective Phosphate Acceptor for Casein Kinase II and Protein Kinase C, Detected in the Cytosolic Fraction of Xenopus laevis Oocytes1

Casein kinase II (CKII) is one of several protein kinases that become activated before germinal-vesicle breakdown in maturing sea-star oocytes. Echinoderm CKII was purified over 11,000-fold with a recovery of approximately 10% by sequential fractionation of the oocyte cytosol on tyrosine-agarose, heparin-agarose, casein-agarose and MonoQ. The purified enzyme contained 45, 38 and 28 kDa polypeptides, which corresponded to its alpha, alpha' and beta subunits respectively. The beta-subunit was autophosphorylated on one major tryptic peptide on serine residues, whereas the alpha'-subunit incorporated phosphate into at least two tryptic peptides primarily on threonine residues. Western-blotting analysis of sea-star oocyte extracts with two different anti-peptide antibodies that recognized conserved regions of the alpha-subunit indicated that the protein levels of the alpha- and alpha'-subunits of CKII were unchanged during oocyte maturation. The purified CKII was partly inactivated (by 25%) by preincubation with protein-serine/threonine phosphatase 2A, but protein-tyrosine phosphatases had no effect. The beta-subunit of CKII was phosphorylated on a serine residue(s) up to 0.54 mol of P/mol of beta-subunit by purified protein kinase C, and this correlated with a 1.5-fold enhancement of its phosphotransferase activity with phosvitin as a substrate. CKII was not a substrate for the maturation-activated myelin basic protein kinase p44mpk from sea-star oocytes, nor for cyclic-AMP-dependent protein kinase. These studies point to possible regulation of CKII by protein phosphorylation.

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Mono-(2-ethylhexyl) Phthalate Rapidly Increases Celsr2 Protein Phosphorylation in HeLa Cells via Protein Kinase C and Casein Kinase 1

Toxicological Sciences ◽

10.1093/toxsci/kfj135 ◽

2006 ◽

Vol 91 (1) ◽

pp. 255-264 ◽

Cited By ~ 8

Author(s):

Stephanie A. Lahousse ◽

Stephanie A. Beall ◽

Kamin J. Johnson

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Protein Phosphorylation ◽

Hela Cells ◽

Casein Kinase ◽

Casein Kinase 1 ◽

Kinase C ◽

Ethylhexyl Phthalate

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Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis.

Cell Regulation ◽

10.1091/mbc.1.3.315 ◽

1990 ◽

Vol 1 (3) ◽

pp. 315-326 ◽

Cited By ~ 51

Author(s):

W M Bement ◽

D G Capco

Keyword(s):

Cell Cycle ◽

Protein Kinase C ◽

Protein Kinase ◽

Xenopus Laevis ◽

Mitotic Cell ◽

Cortical Granule ◽

Mitotic Cell Cycle ◽

Cleavage Furrow ◽

Kinase C ◽

Cortical Granule Exocytosis

Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle.

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Protein kinase C activation antagonizes melatonin-induced pigment aggregation in Xenopus laevis melanophores.

The Journal of Cell Biology ◽

10.1083/jcb.119.6.1515 ◽

1992 ◽

Vol 119 (6) ◽

pp. 1515-1521 ◽

Cited By ~ 46

Author(s):

D Sugden ◽

S J Rowe

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Xenopus Laevis ◽

Pigment Granule ◽

Melatonin Receptors ◽

Kinase C ◽

Pigment Granules ◽

Slow Aggregation ◽

Pigment Aggregation ◽

Induced Aggregation

The pineal hormone, melatonin (5-methoxy N-acetyltryptamine) induces a rapid aggregation of melanin-containing pigment granules in isolated melanophores of Xenopus laevis. Treatment of melanophores with activators of protein kinase C (PKC), including phorbol esters, mezerein and a synthetic diacylglycerol, did not affect pigment granule distribution but did prevent and reverse melatonin-induced pigment aggregation. This effect was blocked by an inhibitor of PKC, Ro 31-8220. The inhibitory effect was not a direct effect on melatonin receptors, per se, as the slow aggregation induced by a high concentration of an inhibitor of cyclic AMP-dependent protein kinase (PKA), adenosine 3',5'-cyclic monophosphothioate, Rp-diastereomer (Rp-cAMPS), was also reversed by PKC activation. Presumably activation of PKC, like PKA activation, stimulates the intracellular machinery involved in the centrifugal translocation of pigment granules along microtubules. alpha-Melanocyte stimulating hormone (alpha-MSH), like PKC activators, overcame melatonin-induced aggregation but this response was not blocked by the PKC inhibitor, Ro 31-8220. This data indicates that centrifugal translocation (dispersion) of pigment granules in Xenopus melanophores can be triggered by activation of either PKA, as occurs after alpha-MSH treatment, or PKC. The very slow aggregation in response to inhibition of PKA with high concentrations of Rp-cAMPS, suggests that the rapid aggregation in response to melatonin may involve multiple intracellular signals in addition to the documented Gi-mediated inhibition of adenylate cyclase.

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