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Published on: October 30, 2013
Purification and characterization of a protein kinase from Xenopus eggs highly specific for ribosomal protein S6
Abstract:
In Xenopus oocytes ribosomal protein S6 becomes phosphorylated on serine residues in response to hormones or growth factors and following microinjection of the tyrosine-specific protein kinases associated with Rous sarcoma virus or Abelson murine leukemia virus. To begin characterization of the enzymes responsible for S6 phosphorylation in this system, we have undertaken the purification of S6 protein kinases from unfertilized Xenopus eggs. DEAE-Sephacel chromatography of crude extracts revealed two peaks of S6 kinase activity, and the peak eluting at 160 mM NaCl was chosen for further purification. Successive chromatography on Mono S, Sephacryl S-200, Mono Q, and heparin-Sepharose resulted in purification of the enzyme to a single protein migrating at Mr = 92,000 on polyacrylamide gels. The final preparation was purified about 500-fold from the DEAE-Sephacel peak with a recovery of 10%. Apparent Km values of the enzyme for ATP and 40 S subunits were 28 and 5 microM, respectively, and the specific activity with 330 microM ATP and 5.6 microM 40 S subunits was 300 nmol/min/mg. The enzyme was inhibited by beta-glycerophosphate, sodium fluoride, potassium phosphate, ADP, heparin, quercetin, and spermine. The availability of a purified S6 protein kinase should facilitate elucidation of the molecular mechanism of S6 phosphorylation during growth stimulation.
Insights
Researchers purified a key enzyme, S6 protein kinase, from Xenopus eggs. This purified kinase is crucial for understanding how ribosomal protein S6 phosphorylation is regulated during cell growth.
Area of Science:
- Molecular and Cellular Biology
- Biochemistry
- Xenopus Oocyte Research
Background:
- Ribosomal protein S6 phosphorylation is a key event in cellular growth and development, often triggered by hormones and growth factors.
- Specific tyrosine-kinases, like those from Rous sarcoma virus, can induce S6 phosphorylation in Xenopus oocytes, indicating the involvement of protein kinases.
- Understanding the endogenous enzymes responsible for S6 phosphorylation in Xenopus is essential for dissecting growth factor signaling pathways.
Purpose of the Study:
- To initiate the characterization of enzymes responsible for S6 protein phosphorylation in Xenopus oocytes.
- To purify the S6 protein kinase from unfertilized Xenopus eggs for detailed enzymatic analysis.
Main Methods:
- Crude extracts from unfertilized Xenopus eggs were subjected to ion-exchange chromatography (DEAE-Sephacel).
- Further purification involved multiple chromatographic steps including Mono S, Sephacryl S-200, Mono Q, and heparin-Sepharose.
- Enzyme kinetics were assessed by determining apparent Km values for ATP and 40 S ribosomal subunits, and specific activity was measured.
Main Results:
- Two peaks of S6 kinase activity were detected, with one eluting at 160 mM NaCl selected for purification.
- A single protein of Mr = 92,000 was purified approximately 500-fold with 10% recovery.
- The purified enzyme exhibited apparent Km values of 28 µM for ATP and 5 µM for 40 S subunits, and was inhibited by various compounds including beta-glycerophosphate and quercetin.
Conclusions:
- A highly purified S6 protein kinase from Xenopus eggs has been obtained.
- The availability of this purified enzyme provides a critical tool for elucidating the molecular mechanisms underlying S6 phosphorylation in response to growth stimulation.
- Further studies using the purified kinase will help unravel signaling pathways involved in cellular growth regulation.
