Small molecule gated split-tyrosine phosphatases and orthogonal split-tyrosine kinases

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Signaling

Background:

• Protein kinases and phosphatases reversibly control cell signaling pathways through phosphorylation and dephosphorylation.
• Existing methods for controlling these enzymes are limited in precision and orthogonality.
• Split protein systems offer a way to conditionally activate enzymes.

Purpose of the Study:

• To design and validate split-tyrosine phosphatases (PTPs) and split-tyrosine kinases (PTKs) controllable by small molecules.
• To demonstrate orthogonal control using different chemical inducers of dimerization (CIDs).
• To establish a methodology for post-translational, orthogonal small molecule control over these enzymes in mammalian cells.

Main Methods:

• Rational design of split-PTPs and split-PTKs fused to FKBP and FRB domains.
• Activation of split enzymes using rapamycin, abscisic acid, and gibberellic acid as orthogonal CIDs.
• Validation of enzyme activity modulation in mammalian cells.

Main Results:

• Successfully designed and validated three split-tyrosine phosphatases (PTPs) controllable by rapamycin.
• Demonstrated orthogonal control of split-tyrosine kinases (PTKs) using abscisic acid and gibberellic acid.
• Showcased activation of both designed split-phosphatases and split-kinases by orthogonal CIDs in mammalian cells.

Conclusions:

• A novel methodology enables post-translational, orthogonal small molecule control over user-defined split-PTKs and split-PTPs.
• This approach allows for precise interrogation and redesign of phosphorylation-dependent signaling pathways.
• The developed system has significant potential for advancing research in cell signaling and developing new therapeutic strategies.