RESUMEN
The activity of protein kinases are naturally gated by a variety of physiochemical inputs, such as phosphorylation, metal ions, and small molecules. In order to design protein kinases that can be gated by user-defined inputs, we describe a sequence dissimilarity based approach for identifying sites in protein kinases that accommodate 25-residue loop insertion while retaining catalytic activity. We further demonstrate that the successful loop insertion mutants provide guidance for the dissection of protein kinases into two fragments that cannot spontaneously assemble and are thus inactive but can be converted into ligand-gated catalytically active split-protein kinases. We successfully demonstrate the feasibility of this approach with Lyn, Fak, Src, and PKA, which suggests potential generality.
Asunto(s)
Mutagénesis Insercional , Proteínas Quinasas/química , Proteínas Quinasas/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteína-Tirosina Quinasas de Adhesión Focal/química , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia , Familia-src Quinasas/química , Familia-src Quinasas/genéticaRESUMEN
Protein kinases phosphorylate client proteins, while protein phosphatases catalyze their dephosphorylation and thereby in concert exert reversible control over numerous signal transduction pathways. We have recently reported the design and validation of split-protein kinases that can be conditionally activated by an added small molecule chemical inducer of dimerization (CID), rapamycin. Herein, we provide the rational design and validation of three split-tyrosine phosphatases (PTPs) attached to FKBP and FRB, where catalytic activity can be modulated with rapamycin. We further demonstrate that the orthogonal CIDs, abscisic acid and gibberellic acid, can be used to impart control over the activity of split-tyrosine kinases (PTKs). Finally, we demonstrate that designed split-phosphatases and split-kinases can be activated by orthogonal CIDs in mammalian cells. In sum, we provide a methodology that allows for post-translational orthogonal small molecule control over the activity of user defined split-PTKs and split-PTPs. This methodology has the long-term potential for both interrogating and redesigning phosphorylation dependent signaling pathways.
Asunto(s)
Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Quinasas/química , Dimerización , Células HEK293 , Humanos , Modelos Moleculares , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismoRESUMEN
The over 500 human protein kinases are estimated to phosphorylate at least one-third of the proteome. This posttranslational modification is of paramount importance to intracellular signaling and its deregulation is linked to numerous diseases. Deciphering the specific cellular role of a protein kinase of interest remains challenging given their structural similarity and potentially overlapping activity. In order to exert control over the activity of user-defined kinases and allow for understanding and engineering of complex signal transduction pathways, we have designed ligand inducible split protein kinases. In this approach, protein kinases are dissected into two fragments that cannot spontaneously assemble and are thus inactive. The two kinase fragments are attached to chemical inducers of dimerization (CIDs) that allow for ligand induced heterodimerization and concomitant activation of kinase activity.