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...

Small-molecule-mediated rescue of protein function by an inducible proteolytic shunt

“…SURF represents an attractive alternative when the protein of interest is known to take part in protein-protein interactions or multimeric complexes as rescue is inextricably linked to generation of the native protein. In the future this cleavage-dependent rescue technology could be expanded to include other methods of protein regulation such as mislocalization or autoinhibition (34).…”

Directed Evolution of a Small-Molecule-Triggered Intein with Improved Splicing Properties in Mammalian Cells

“…Although natural inteins splice spontaneously, inteins that undergo splicing in a small-molecule-dependent manner have been developed by fusing intein halves with proteins that dimerize in the presence of a small molecule (Mootz and Muir, 2002; Mootz et al, 2003; Shi and Muir, 2005), or by directed evolution in which a library of intact inteins fused to a ligand-binding domain was screened to splice in the presence, but not the absence, of a small molecule (Buskirk et al, 2004). These small-molecule-dependent inteins have enabled protein function in cells to be controlled post-translationally by the addition of an exogenous, cell-permeable molecule (Mootz and Muir, 2002, Mootz et al, 2003, Buskirk et al, 2004; Mootz et al, 2004; Shi and Muir, 2005; Yuen et al, 2006; Schwartz et al, 2007; Hartley and Madhani, 2009). …”

Studying protein structure and function using semisynthesis

“…This has allowed the development of so-called conditional protein splicing systems in which the splicing reaction is triggered by induced proximity of the intein fragments through either the use of small molecule or light stimulation. [31][32][33] We have shown that conditional protein splicing can be used to for the rapid and tunable activation of proteins in a variety of cell types 34 and even in a living animal. 35 …”