Controlling the Activity of the Tec Kinase Itk by Mutation of the Phenylalanine Gatekeeper Residue

Abstract

The regulatory spine is a set of conserved residues that are assembled and disassembled upon activation and inactivation of kinases. We recently identified the regulatory spine within the immunologically important Tec family kinases and have shown that in addition to the core spine residues within the kinase domain itself, contributions from the SH2-kinase linker region result in an extended spine structure for this kinase family. Disruption of the regulatory spine, either by mutation or removal of the amino-terminal SH2-kinase linker region or mutation of core spine residues, leads to inactivation of the Tec kinases. With a focus on the Tec family members, Itk and Btk, we now show that the gatekeeper residue is also critical for the assembly of the regulatory spine. Mutation of the bulky Itk F434 gatekeeper residue to alanine or glycine inactivates Itk. The activity of the Itk F434A mutant can be recovered by a secondary-site mutation within the N-terminal lobe, specifically L432I. The Itk L432I mutation likely rescues the activity of the gatekeeper F434A mutation by promoting the assembly of the regulatory spine. We also show that mutation of the Itk and Btk gatekeeper residues to methionine is sufficient to activate the isolated kinase domains of Tec kinases in the absence of the amino-terminal SH2-kinase linker. Thus, shifting the conformational equilibrium between the assembled and disassembled states of the regulatory spine by changing the nature of the gatekeeper residue is key to regulating the activity of Tec kinases.

Deciphering the Structural Basis of Eukaryotic Protein Kinase Regulation

“…Sh1 is also capable of maintaining some catalytic activity in the absence of RS3 by completing the R-spine through the gatekeeper residue (Sh2). Previous studies showed that Sh2 and Sh3 are equally important for catalytic activity because either residue has the ability to compensate for the absence of the other, as in IL2-inducible T-cell kinase (Itk), and that at least one was mandatory for maintaining catalytic activity [25]. Here we confirm that the absence of Sh3 and Sh2 in PKA abolishes catalytic activity and returning either one enables the partial rescue of catalytic function.…”

A Conserved Isoleucine Maintains the Inactive State of Bruton's Tyrosine Kinase

“…Striking examples are the Tec (7–10) and Src family tyrosine kinases (11, 12); these kinase families are closely related evolutionarily but employ opposing regulatory schemes. Tec kinase domains, even when phosphorylated on the activation loop, are inactive, relying on direct association with regulatory regions outside of the kinase domain to achieve activation (13–16). In contrast, phosphorylated Src family kinase domains are fully active by themselves (17, 18), and are inhibited by association with their regulatory domains (19, 20).…”

Design principles underpinning the regulatory diversity of protein kinases

“…Based on the LSP method, Kornev et al [49,50] defined some of the hydrophobic network residues as the ‘regulatory spine’ because they observed that the hydrophobic network is assembled in active kinases, but disassembled in the inactive forms (figure 3). Consistent with the regulatory role for the hydrophobic network, mutation of the spine residue resulted in kinase inactivation in some tyrosine kinases [51,52]. The conservation of the hydrophobic network in ELKs suggests that it performs a similar regulatory role; however, this hypothesis needs to be tested through structural and biochemical studies.…”