J. Michael Bradshaw

The Src, Syk, and Tec family kinases: distinct types of molecular switches.

The Src, Syk, and Tec family kinases are three of the most well characterized tyrosine kinase families found in the human genome. Members of these kinase families function downstream of antigen and F(c) receptors in hematopoietic cells and transduce signals leading to calcium mobilization, altered gene expression, cytokine production, and cell proliferation. Over the last several years, structural and biochemical studies have begun to uncover the molecular mechanisms regulating activation of these kinases. It appears that each kinase family functions as a distinct type of molecular switch. This review discusses the activation of the Src, Syk, and Tec kinases from the perspective of structure, phosphorylation, allosteric regulation, and kinetics. The multiple factors that regulate the Src, Syk, and Tec families illustrate the important role played by each of these kinases in immune cell signaling.

Molecular Mechanism of the Syk Activation Switch

Many immune signaling pathways require activation of the Syk tyrosine kinase to link ligation of surface receptors to changes in gene expression. Despite the central role of Syk in these pathways, the Syk activation process remains poorly understood. In this work we quantitatively characterized the molecular mechanism of Syk activation in vitro using a real time fluorescence kinase assay, mutagenesis, and other biochemical techniques. We found that dephosphorylated full-length Syk demonstrates a low initial rate of substrate phosphorylation that increases during the kinase reaction due to autophosphorylation. The initial rate of Syk activity was strongly increased by either pre-autophosphorylation or binding of phosphorylated immune tyrosine activation motif peptides, and each of these factors independently fully activated Syk. Deletion mutagenesis was used to identify regions of Syk important for regulation, and residues 340–356 of the SH2 kinase linker region were identified to be important for suppression of activity before activation. Comparison of the activation processes of Syk and Zap-70 revealed that Syk is more readily activated by autophosphorylation than Zap-70, although both kinases are rapidly activated by Src family kinases. We also studied Syk activity in B cell lysates and found endogenous Syk is also activated by phosphorylation and immune tyrosine activation motif binding. Together these experiments show that Syk functions as an “OR-gate” type of molecular switch. This mechanism of switch-like activation helps explain how Syk is both rapidly activated after receptor binding but also sustains activity over time to facilitate longer term changes in gene expression.

Activation Mechanism and Steady State Kinetics of Bruton's Tyrosine Kinase

Brutons tyrosine kinase (BTK) is a member of the Tec non-receptor tyrosine kinase family that is involved in regulating B cell proliferation. To better understand the enzymatic mechanism of the Tec family of kinases, the kinetics of BTK substrate phosphorylation were characterized using a radioactive enzyme assay. We first examined whether autophosphorylation regulates BTK activity. Western blotting with a phosphospecific antibody revealed that BTK rapidly autophosphorylates at Tyr551 within its activation loop in vitro. Examination of a Y551F BTK mutant indicated that phosphorylation of Tyr551 causes a 10-fold increase in BTK activity. We then proceeded to characterize the steady state kinetic mechanism of BTK. Varying the concentrations of ATP and S1 peptide (biotin-Aca-AAAEEIY-GEI-NH2) revealed that BTK employs a ternary complex mechanism with KmATP = 84 ± 20 μm and KmS1 = 37 ± 8 μm. Inhibition studies were also performed to examine the order of substrate binding. The inhibitors ADP and staurosporine were both found to be competitive with ATP and non-competitive with S1, indicating binding of ATP and S1 to BTK is either random or ordered with ATP binding first. Negative cooperativity was also found between the S1 and ATP binding sites. Unlike ATP site inhibitors, substrate analog inhibitors did not inhibit BTK at concentrations less than 1 mm, suggesting that BTK may employ a “substrate clamping” type of kinetic mechanism whereby the substrate Kd is weaker than Km. This investigation of BTK provides the first detailed kinetic characterization of a Tec family kinase.

Role of the N- and C-lobes of calmodulin in the activation of Ca(2+)/calmodulin-dependent protein kinase II.

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.

A fluorescence resonance energy transfer-based binding assay for characterizing kinase inhibitors: Important role for C-terminal biotin tagging of the kinase

Novel biochemical strategies are needed to identify the next generation of protein kinase inhibitors. One promising new assay format is a competition binding approach that employs time-resolved fluorescence resonance energy transfer (TR-FRET). In this assay, a FRET donor is bound to the kinase via a purification tag, whereas a FRET acceptor is bound via a tracer-labeled inhibitor. Displacement of the tracer by an unlabeled inhibitor eliminates FRET between the fluorophores and provides a readout on binding. Although promising, this technique has so far been limited in applicability in part by a lack of signal strength is some cases and also by an inability to predict whether a particular tagging strategy will show robust FRET. In this work, we sought to better understand the factors that give rise to a strong FRET signal in this assay. We determined the magnitude of FRET for several tyrosine kinases using different purification tags (biotin, glutathione S-transferase [GST], and His) placed at either the N terminus or C terminus of the kinase. It was observed that coupling the FRET acceptor to the kinase C terminus using a biotin/streptavidin interaction resulted in the greatest increase in FRET. Specifically, for multiple kinases, the signal/background ratio was at least 3-fold better using C-terminal biotinylation compared with tagging at the N terminus using a His/anti-His antibody or GST/anti-GST antibody interaction. In one case, the FRET signal using C-terminal biotin tagging was more than 150-fold over background. This strong FRET signal facilitated development of improved inhibitor binding assays that required only tens of picomolar enzyme or tracer-labeled inhibitor. Together, these results indicate that C-terminal biotinylation is a promising tagging strategy for developing an optimal FRET-based competition binding assay for tyrosine kinases.

The irreversible covalent fibroblast growth factor receptor inhibitor PRN1371 exhibits sustained inhibition of FGFR after drug clearance

An increasing number of cancers are known to harbor mutations, translocations, or amplifications in the fibroblast growth factor receptor (FGFR) family of kinases. The FGFR inhibitors evaluated in clinical trials to date have shown promise at treating these cancers. Here, we describe PRN1371, an irreversible covalent inhibitor of FGFR1-4 targeting a cysteine within the kinase active site. PRN1371 demonstrated strong FGFR potency and excellent kinome-wide selectivity in a number of biochemical and cellular assays, including in various cancer cell lines exhibiting FGFR alterations. Furthermore, PRN1371 maintained FGFR inhibition in vivo, not only when circulating drug levels were high but also after the drug had been cleared from circulation, indicating the possibility of sustained FGFR inhibition in the clinic without the need for continuous drug exposure. Durable tumor regression was also obtained in multiple tumor xenografts and patient-derived tumor xenograft models and was sustained even using an intermittent dosing strategy that provided drug holidays. PRN1371 is currently under clinical investigation for treatment of patients with solid tumors. Mol Cancer Ther; 16(12); 2668–76. ©2017 AACR.

Co-inhibition of immunoproteasome subunits LMP2 and LMP7 is required to block autoimmunity

Cells of hematopoietic origin express high levels of the immunoproteasome, a cytokine‐inducible proteasome variant comprising the proteolytic subunits LMP2 (β1i), MECL‐1 (β2i), and LMP7 (β5i). Targeting the immunoproteasome in pre‐clinical models of autoimmune diseases with the epoxyketone inhibitor ONX 0914 has proven to be effective. ONX 0914 was previously described as a selective LMP7 inhibitor. Here, we show that PRN1126, developed as an exclusively LMP7‐specific inhibitor, has limited effects on IL‐6 secretion, experimental colitis, and experimental autoimmune encephalomyelitis (EAE). We demonstrate that prolonged exposure of cells with ONX 0914 leads to inhibition of both LMP7 and LMP2. Co‐inhibition of LMP7 and LMP2 with PRN1126 and LMP2 inhibitors LU‐001i or ML604440 impairs MHC class I cell surface expression, IL‐6 secretion, and differentiation of naïve T helper cells to T helper 17 cells, and strongly ameliorates disease in experimental colitis and EAE. Hence, co‐inhibition of LMP2 and LMP7 appears to be synergistic and advantageous for the treatment of autoimmune diseases.

The Bruton's tyrosine kinase inhibitor PRN473 inhibits neutrophil recruitment via inhibition of Mac-1 signaling

Following inflammatory stimuli, neutrophils are recruited to sites of inflammation and exert effector functions that often have deleterious effects on tissue integrity, which can lead to organ failure. Brutons tyrosine kinase (Btk) is expressed in neutrophils and constitutes a promising pharmacological target for neutrophil‐mediated tissue damage. Here, we evaluate a selective reversible inhibitor of Btk, PRN473, for its ability to dampen neutrophil influx via inhibition of adhesion receptor signalling pathways.