Related Experiment Videos
Activation of messenger-independent protein kinases in wild-type and phorbol ester-resistant EL4 thymoma cells
K E Meier1, K A Licciardi, T A Haystead
1Department of Pharmacology, University of Washington, Seattle 98195.
Abstract:
Phorbol esters, acting via activation of the protein kinase C family of protein serine/threonine kinases, are able to exert profound effects on various cellular functions. In this study, we used the EL4 thymoma cell line to study the potential role of "downstream" protein serine/threonine kinases in cellular responses to phorbol esters. In wild-type EL4 cells, addition of phorbol ester caused a rapid activation of kinase activity toward RRLSSLRA (S6P). This increased activity was maintained for at least 15 min but diminished to control levels by 60 min. Activation of a myelin basic protein (MBP) kinase was also seen in response to phorbol ester. In a variant EL4 cell line in which phorbol ester does not induce interleukin 2 transcription, phorbol ester failed to activate either the S6P kinase or MBP kinase. Partial purification of the activated S6P and MBP kinases from wild-type cells showed that they represent separate enzymes that are distinct from protein kinase C. Although the variant cells had reduced levels of protein kinase C as compared with the wild-type cells, the amount of membrane-bound enzyme increased in response to phorbol 12-myristate 13-acetate in both wild-type and variant cells. Treatment of intact cells with phorbol ester resulted in phosphorylation of some of the same protein substrates in both cell lines. Okadaic acid, a phosphatase inhibitor, increased S6P and MBP kinase activities in both wild-type and variant cells. Thus, phorbol ester failed to activate the S6P and MBP kinases in the variant cells even though these cells express activatable protein kinase C, S6P kinase, and MBP kinase. Two protein kinase inhibitors, staurosporine and H-7, inhibited the activity of all three kinases in vitro, while a peptide inhibitor (PKC 19-31) showed specificity for protein kinase C. In summary, these results suggest that activation of messenger-independent protein kinases may be critical for certain protein kinase C-dependent responses.
Insights
Phorbol esters activate protein kinase C (PKC), influencing cellular functions. This study reveals that downstream kinases, S6P and MBP kinase, are crucial for PKC-dependent responses, even in cells with impaired interleukin 2 transcription.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Phorbol esters activate protein kinase C (PKC), a family of serine/threonine kinases, impacting diverse cellular functions.
- Understanding the downstream signaling pathways activated by PKC is crucial for elucidating cellular responses.
Purpose of the Study:
- To investigate the role of downstream protein serine/threonine kinases in cellular responses to phorbol esters.
- To characterize the activation of S6P kinase and myelin basic protein (MBP) kinase in response to phorbol ester stimulation.
Main Methods:
- Utilized EL4 thymoma cell lines (wild-type and a variant) to study phorbol ester-induced kinase activation.
- Employed RRLSSLRA peptide and myelin basic protein (MBP) as substrates to assay S6P kinase and MBP kinase activity, respectively.
- Performed partial purification of kinases and used specific inhibitors (staurosporine, H-7, PKC 19-31) and a phosphatase inhibitor (okadaic acid).
Main Results:
- Phorbol ester rapidly activated S6P kinase and MBP kinase in wild-type EL4 cells, with activation sustained for at least 15 minutes.
- In a variant EL4 cell line unable to transcribe interleukin 2 in response to phorbol ester, neither S6P kinase nor MBP kinase activation was observed.
- Activated S6P and MBP kinases were distinct from PKC, and while variant cells had lower PKC levels, membrane-bound PKC increased similarly in both cell types upon phorbol ester treatment.
- Okadaic acid enhanced S6P and MBP kinase activities in both cell lines, and protein kinase inhibitors affected kinase activities in vitro.
Conclusions:
- Activation of messenger-independent protein kinases, specifically S6P and MBP kinases, appears critical for certain protein kinase C-dependent cellular responses.
- The observed defect in S6P and MBP kinase activation in the variant cells suggests a critical role for these downstream kinases in mediating phorbol ester-induced cellular events, independent of direct PKC activity levels.