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Transcription factor phosphorylation by pp90(rsk2). Identification of Fos kinase and NGFI-B kinase I as pp90(rsk2)
K D Swanson1, L K Taylor, L Haung
1Alzheimer's Research Laboratory, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Abstract:
The in vitro phosphorylation of transcription factors by growth factor-activated protein kinases has resulted in the discovery of a number of activities whose identities and relationships to one another are unclear. Fos kinase is a growth factor-stimulated serine/threonine protein kinase that phosphorylates c-Fos at serine 362 within the carboxyl-terminal regulatory domain. Fos kinase activation is dependent on p21(ras) and mitogen-activated protein kinase/ERK kinase kinase (MEK) activity and is independent of phosphatidylinositol 3-kinase activity. We have purified Fos kinase by affinity chromatography using the Sepharose-linked protein kinase inhibitor, bisindolylmaleimide (BIM). Fos kinase has an apparent molecular mass of 88 kDa, and mass spectrophotometric analysis of the isolated protein showed that it produced tryptic fragments identical to those predicted for pp90(rsk2). Fos kinase isolated from nerve growth factor-stimulated PC12 cells is indistinguishable from NGFI-B kinase I, based on their chromatographic behavior, substrate specificities, and relative sensitivity to BIM. Furthermore, we have distinguished Fos kinase from calcium/cAMP response element-binding protein (CREB) kinase. Therefore, Fos kinase and NGFI-B kinase I and pp90(rsk2) represent the same protein kinase species. Moreover, we report that pp90(rsk2) exists within nerve growth factor-stimulated PC12 cells as two chromatographically and immunologically distinct species. Finally, we demonstrate that CREB kinase is distinct from pp90(rsk2).
Insights
Fos kinase, a growth factor-stimulated protein kinase, phosphorylates c-Fos. This study identifies Fos kinase as pp90(rsk2), distinct from CREB kinase.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Growth factor-activated protein kinases play crucial roles in cellular signaling pathways.
- The precise identities and relationships of these kinases, particularly those phosphorylating transcription factors like c-Fos, remain incompletely understood.
- Fos kinase, a serine/threonine protein kinase, is activated by growth factors and phosphorylates c-Fos.
Purpose of the Study:
- To identify and characterize Fos kinase, a growth factor-stimulated protein kinase.
- To elucidate the relationship between Fos kinase, pp90(rsk2), NGFI-B kinase I, and CREB kinase.
- To investigate the different forms of pp90(rsk2) present in nerve growth factor-stimulated PC12 cells.
Main Methods:
- Purification of Fos kinase using affinity chromatography with a bisindolylmaleimide (BIM)-Sepharose resin.
- Mass spectrophotometric analysis of tryptic fragments to determine protein identity.
- Comparative analysis of chromatographic behavior, substrate specificities, and BIM sensitivity.
- Immunological characterization of pp90(rsk2) species.
Main Results:
- Fos kinase was purified and identified as pp90(rsk2) based on molecular mass and tryptic fragment analysis.
- Fos kinase activity is dependent on p21(ras) and MEK, but not phosphatidylinositol 3-kinase.
- Purified Fos kinase was indistinguishable from NGFI-B kinase I, confirming they are the same protein kinase species.
- pp90(rsk2) was found to exist as two distinct species in nerve growth factor-stimulated PC12 cells.
- CREB kinase was demonstrated to be distinct from pp90(rsk2).
Conclusions:
- Fos kinase is identical to pp90(rsk2) and NGFI-B kinase I.
- pp90(rsk2) is a key player in growth factor signaling pathways, phosphorylating c-Fos.
- Distinct forms of pp90(rsk2) exist, and CREB kinase is a separate entity.
- This research clarifies the identity and relationships of important protein kinases involved in cellular regulation.