Cleavage Arrest of Early Frog Embryos by the G Protein-activated Protein Kinase PAK I

Regina D Rooney,Polygena T Tuazon,William E Meek,Edward J Carroll,Jeffrey J Hagen,Edwin L Gump,Curtis A Monnig,Tracy Lugo,Jolinda A Traugh

doi:10.1074/jbc.271.35.21498

Regina D Rooney, Polygena T Tuazon + Show 7 more

Open Access

https://doi.org/10.1074/jbc.271.35.21498

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Abstract

PAK I is a member of the PAK (p21-activated protein kinase) family and is activated by Cdc42 (Jakobi, R., Chen, C.-J., Tuazon, P. T., and Traugh, J. A. (1996) J. Biol. Chem. 271, 6206-6211). To examine the effects of PAK I on cleavage arrest, subfemtomole amounts of endogenously active (58 kDa) and inactive (60 kDa) PAK I and a tryptic peptide (37 kDa) containing the active catalytic domain were injected into one blastomere of 2-cell frog embryos. Active PAK I resulted in cleavage arrest in the injected blastomere at mitotic metaphase, whereas the uninjected blastomere progressed through mid- to late cleavage. Injection of other protein kinases at similar concentrations had no effect on cleavage. Endogenous PAK I was highly active in frog oocytes, and antibody to PAK I reacted specifically with protein of 58-60 kDa. PAK I protein was decreased at 60 min post-fertilization, with little or no PAK I protein or activity detectable at 80 min post-fertilization or in 2-cell embryos. At the 4-cell stage PAK I protein increased, but the protein kinase was present primarily as an inactive form. Rac2 and Cdc42, but not Rac 1, were identified in oocytes and throughout early embryo development. Thus, PAK I appears to be a potent cytostatic protein kinase involved in maintaining cells in a non-dividing state. PAK I activity is high in oocytes and appears to be regulated by degradation/synthesis and through autophosphorylation via binding of Cdc42. PAK I may act through regulation of the stress-activated protein kinase signaling pathway and/or by direct regulation of multiple metabolic pathways.

Highlights

Significant progress has been made in understanding the activation of components in signaling pathways that control cell growth and differentiation in vertebrates
There is a large body of research showing that progression through the cell cycle is highly regulated by the maturation promoting factor cascade and the Ras/MAP kinase (MAPK)1 cascade
Injection of c-mos cDNA into one blastomere of 2-cell frog embryos leads to cleavage arrest at metaphase; injection of c-mos antisense RNA reverses the effect [6, 9, 10]

Summary

EXPERIMENTAL PROCEDURES

Materials—Trypsin (diphenylcarbamyl chloride-treated), soybean trypsin inhibitor, and mixed histone IIAS were obtained from Sigma. Protein kinase activity was assayed with H4 (2.0 ␮g) in phosphorylation buffer containing 50 mM Tris-HCl, pH 7.4, l0 mM MgCl2, 30 mM 2-mercaptoethanol, 0.20 mM [␥-32P]ATP (specific activity 1000 cpm/pmol), and PAK I in a final volume of 70 ␮l. To assay for activation of PAK I by CDC42, aliquots (30 ␮l) of column fractions were incubated with the G protein preloaded with GTP␥S or GDP in 70 ␮l of phosphorylation buffer containing 1.4 ␮g of soybean trypsin inhibitor at 30 °C for 10 min, as described by Jakobi et al [30], resulting in autophosphorylation of PAK I. To prepare large amounts of the catalytic domain (p37), inactive PAK I purified through the protamine-agarose step was subjected to limited tryptic digestion for 30 s, followed by addition of a 10-fold excess of soybean trypsin inhibitor [33].

RESULTS

DISCUSSION

Methods