Activation of an Autoregulated Protein Kinase by Conditional Protein Splicing

Abstract

The temporal and spatial control of protein function is of fundamental importance in biology. Most cellular processes require that a small subset of the proteome be active at a particular time and place, and that this activity have a defined duration. To probe the role of a protein in a biological system, one must be able to control these parameters as precisely as possible. [1,2] We recently developed a new tool, termed conditional protein splicing (CPS), to control the primary structure, and hence function, potentially of any protein by using a small molecule. [3,4] The basic principle of CPS is illustrated in Figure 1 a. Exploitation of a split intein that is only active in the presence of the small molecule rapamycin allows proteins or polypeptides to be linked by a peptide bond through protein splicing. The sequences of interest are expressed as recombinant fusions to the N-and C-terminal halves of the intein, which are themselves linked to FKBP and FRB domains. These domains form a high-affinity ternary complex with rapamycin.[5] The induced proximity of the intein halves in this complex mediates the reconstitution of the active intein. Since CPS acts at the posttranslational level, it has the advantage of a short response time (as little as 10 min), which allows high temporal resolution.There are many conceivable strategies for specifically altering the function of a protein by CPS. The most obvious method is to reassemble the protein from two inactive pieces and thereby switch on its activity. This strategy exploits the bond-making feature of CPS and requires that the newly spliced polypeptide spontaneously adopts an active structure. We decided to take advantage of another feature of the CPS reaction, the peptide-bond-breaking steps. The CPS system depicted in Figure 1 a can be regarded not only as a conditional protein ligase that forms the peptide bond between the two extein sequences, but also as a conditional protease that breaks the peptide bonds between the intein and extein sequences. We conceived a strategy in which the protein of interest and a peptide sequence that acts as an inhibitor are fused to opposite ends of one of the CPS constructs such that they are cleaved from one another in the course of the protein splicing reaction. This cleavage should result in a relative increase in the activity of the protein because the inhibitor should display a lower potency when free than while fused to the protein as a result of its higher local concentration in the intramolecular arrangement. This design borrows from the principles often used by nature to control the activity of enzymes. So-called active-site-directed intrasteric autoregulation [6] has been observed for many proteins, for example, zymogens are kept in an autoinhibited state until posttranslational processing reveals the active protease.Protein kinases are key players in a myriad of important processes in the cell and are thus attractive targets upon which to test our idea. We chose the cAMP-dependent protein kinase (PKA) for our investi...

Recent progress in intein research: from mechanism to directed evolution and applications

“…For trans-splicing split inteins the control of the association of the intein fragments was in the focus to generate artificial switches. Referred to as conditional protein splicing (CPS) systems, the rapamycin-binding domains FKBP and FRB were used to bring about proximity and thereby high local concentrations of the split intein fusion constructs to trigger the reconstitution of intein activity [17,[121][122][123][124]. Alternatively, the phytochrome B (PhyB) and the PIF3 phytochrome binding domain (PIF3-APB) served to control intein fragment association with light [125].…”

Control of Transcription Factor Activity and Osteoblast Differentiation in Mammalian Cells Using an Evolved Small-Molecule-Dependent Intein

“…Rapamycin-dependent splicing in this systems has been shown to mediate the joining of two epitope tags in mammalian cells 14 as well as the removal of an inhibitory peptide from a protein kinase A-split intein fusion using purified proteins in solution. 15 Target proteins for which inhibitory peptides are not known or that are not stable when separated and expressed as two intein-fused halves, however, may prove difficult to manipulate using a split intein system. Regulated inteins have not yet been reported to control native protein function in mammalian cells.…”

Synthetic protein switches: design principles and applications

“…In addition, a rapamycin-inducible mutant of protein kinase A (PKA) was generated based on split-inteinmediated excision [74] of an active site-directed competitive inhibitor of PKA ( Figure 4D) [75]. However, proof-ofprinciple is limited to in vitro applications.…”