Shane C. Masters

Sphingosine-dependent Protein Kinase-1, Directed to 14-3-3, Is Identified as the Kinase Domain of Protein Kinase Cδ

Some protein kinases are known to be activated by d-erythro-sphingosine (Sph) or N,N-dimethyl-d-erythro-sphingosine (DMS), but not by ceramide, Sph-1-P, other sphingolipids, or phospholipids. Among these, a specific protein kinase that phosphorylates Ser60, Ser59, or Ser58 of 14-3-3β, 14-3-3η, or 14-3-3ζ, respectively, was termed “sphingosine-dependent protein kinase-1” (SDK1) (Megidish, T., Cooper, J., Zhang, L., Fu, H., and Hakomori, S. (1998) J. Biol. Chem. 273, 21834–21845). We have now identified SDK1 as a protein having the C-terminal half kinase domain of protein kinase Cδ (PKCδ) based on the following observations. (i) Large-scale preparation and purification of proteins showing SDK1 activity from rat liver (by six steps of chromatography) gave a final fraction with an enhanced level of an ∼40-kDa protein band. This fraction had SDK1 activity ∼50,000-fold higher than that in the initial extract. (ii) This protein had ∼53% sequence identity to the Ser/Thr kinase domain of PKCδ based on peptide mapping using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry data. (iii) A search for amino acid homology based on the BLAST algorithm indicated that the only protein with high homology to the ∼40-kDa band is the kinase domain of PKCδ. The kinase activity of PKCδ did not depend on Sph or DMS; rather, it was inhibited by these sphingoid bases, i.e. PKCδ did not display any SDK1 activity. However, strong SDK1 activity became detectable when PKCδ was incubated with caspase-3, which releases the ∼40-kDa kinase domain. PKCδ and SDK1 showed different lipid requirements and substrate specificity, although both kinase activities were inhibited by common PKC inhibitors. The high susceptibility of SDK1 to Sph and DMS accounts for their important modulatory role in signal transduction.

Role of the 14-3-3 C-terminal loop in ligand interaction.

14‐3‐3 proteins are a family of conserved dimeric molecules that interact with a broad range of target proteins, most of which contain phosphoserine/threonine. The amphipathic groove of 14‐3‐3 is the main structural feature involved in mediating its associations. We have studied another domain of 14‐3‐3, the C‐terminal loop, to determine what role it plays in ligand interaction. A truncated form of 14‐3‐3ζ lacking this C‐terminal loop was generated and found to bind with higher affinity than the wild‐type 14‐3‐3ζ protein to the ligands Raf‐1 and Bad. Interestingly, the truncated 14‐3‐3ζ also showed increased association with the 14‐3‐3 binding‐deficient Bad/S136A mutant. Taken together, these data support a role for the C‐terminal loop as a general inhibitor of 14‐3‐3/ligand interactions. This may provide a mechanism by which inappropriate associations with 14‐3‐3 are prevented. Proteins 2002;49:321–325.

Monitoring 14-3-3 Protein Interactions with a Homogeneous Fluorescence Polarization Assay

The 14-3-3 proteins mediate phosphorylation-dependent protein-protein interactions. Through binding to numerous client proteins, 14-3-3 controls a wide range of physiological processes and has been implicated in a variety of diseases, including cancer and neurodegenerative disorders. To better understand the structure and function of 14-3-3 proteins and to develop small-molecule modulators of 14-3-3 proteins for physiological studies and potential therapeutic interventions, the authors have designed and optimized a highly sensitive fluorescence polarization (FP)–based 14-3-3 assay. Using the interaction of 14-3-3 with a fluorescently labeled phosphopeptide from Raf-1 as amodel system, they have achieved a simple 1-step “mixand-measure” method for analyzing 14-3-3 proteins. This is a solution-based, versatile method that can be used tomonitor the binding of 14-3-3with a variety of client proteins. The 14-3-3 FP assay is highly stable and has achieved a robust performance in a 384-well format with a demonstrated signal-to-noise ratio greater than 10 and a Z• factor greater than 0.7. Because of its simplicity and high sensitivity, this assay is generally applicable to studying 14-3-3/client-protein interactions and especially valuable for high-throughput screening of 14-3-3 modulators.

The proapoptotic protein Bad binds the amphipathic groove of 14-3-3ζ

Through interaction with a multitude of target proteins, 14-3-3 proteins participate in the regulation of diverse cellular processes including apoptosis. These 14-3-3-interacting proteins include a proapoptotic Bcl-2 homolog, Bad (Bcl-2/Bcl-XL-associated death promoter). To understand how 14-3-3 interacts with Bad and modulates its function, we have identified structural elements of 14-3-3 necessary for 14-3-3/Bad association. 14-3-3 contains a conserved amphipathic groove that is required for binding to several of its ligands. We used peptides of known binding specificity as competitors to demonstrate that Bad interacts with 14-3-3zeta via its amphipathic groove. More detailed analysis revealed that several conserved residues in the groove, including Lys-49, Val-176, and Leu-220, were critical for Bad interaction. These results were applied to investigations of the ability of 14-3-3 to prevent Bad-induced cell death. When co-expressed with Akt, wild-type 14-3-3 could reduce the ability of Bad to cause death, however 14-3-3zetaK49E, which cannot bind Bad, failed to inhibit Bad. It seems that the amphipathic groove of 14-3-3 represents a general binding site for multiple ligands, raising issues related to competition of ligands for 14-3-3.