Related Experiment Videos
Multisite autophosphorylation of p21-activated protein kinase gamma-PAK as a function of activation
1Department of Biochemistry, University of California, Riverside, California 92521, USA.
Abstract:
p21-activated protein kinase (PAK) is a family of serine/threonine kinases whose activity is stimulated by binding to small G-proteins such as Cdc42 and subsequent autophosphorylation. Focusing on the ubiquitous gamma-isoform of PAK in this study, baculovirus-infected insect cells were used to obtain recombinant gamma-PAK, while native gamma-PAK was isolated from rabbit reticulocytes. Two-dimensional gel electrophoresis of gamma-PAK followed by immunoblot analysis revealed a similar profile for native and recombinant gamma-PAK, both consisting of multiple protein spots. Following Cdc42-stimulated autophosphorylation, the two-dimensional profiles of native and recombinant gamma-PAK were characterized by a similar acidic shift, suggesting a common response to Cdc42. To understand the effect of differential phosphorylation on its activation status, gamma-PAK autophosphorylation was conducted in the presence or absence of activators such as Cdc42 and histone II-AS, followed by tryptic digestion and comparative two-dimensional phosphopeptide mapping. The major phosphopeptides were subjected to a combination of manual and automated amino acid sequencing. Overall, eight autophosphorylation sites were identified in Cdc42-activated gamma-PAK, six of which are in common with those previously reported in alpha-PAK, while Ser-19 and Ser-165 appear to be uniquely phosphorylated in the gamma-form. Further, the phosphorylation of Ser-141, Ser-165, and Thr-402 was found to correlate with gamma-PAK activation.
Insights
p21-activated protein kinase (PAK) gamma-isoform activation was studied using recombinant and native forms. Cdc42 binding and autophosphorylation revealed key phosphorylation sites, including unique sites for gamma-PAK, correlating with kinase activation.
Area of Science:
- Molecular Biology
- Protein Kinase Research
Background:
- p21-activated protein kinase (PAK) is a family of serine/threonine kinases activated by small G-proteins like Cdc42.
- The gamma-isoform of PAK is ubiquitously expressed and its activation mechanism requires further elucidation.
Purpose of the Study:
- To characterize the activation status and phosphorylation sites of the gamma-PAK isoform.
- To compare the behavior of native and recombinant gamma-PAK.
- To identify specific phosphorylation sites critical for gamma-PAK activation.
Main Methods:
- Production of recombinant gamma-PAK using baculovirus-infected insect cells and isolation of native gamma-PAK from rabbit reticulocytes.
- Two-dimensional gel electrophoresis and immunoblot analysis to assess protein profiles.
- Cdc42-stimulated autophosphorylation, tryptic digestion, and two-dimensional phosphopeptide mapping.
- Manual and automated amino acid sequencing of phosphopeptides.
Main Results:
- Native and recombinant gamma-PAK exhibited similar profiles on two-dimensional gels.
- Cdc42-stimulated autophosphorylation induced a common acidic shift in both forms.
- Eight autophosphorylation sites were identified in activated gamma-PAK, with Ser-19 and Ser-165 being unique to the gamma-isoform.
- Phosphorylation of Ser-141, Ser-165, and Thr-402 correlated with gamma-PAK activation.
Conclusions:
- The study provides a detailed characterization of gamma-PAK phosphorylation sites and their role in activation.
- Unique phosphorylation sites in gamma-PAK suggest isoform-specific regulatory mechanisms.
- The findings contribute to understanding the molecular basis of PAK signaling pathways.