Discovery of an exosite on the SOCS2-SH2 domain that enhances SH2 binding to phosphorylated ligands

Suppressor of cytokine signaling (SOCS)2 protein is a key negative regulator of the growth hormone (GH) and Janus kinase (JAK)-Signal Transducers and Activators of Transcription (STAT) signaling cascade. The central SOCS2-Src homology 2 (SH2) domain is characteristic of the SOCS family proteins and is an important module that facilitates recognition of targets bearing phosphorylated tyrosine (pTyr) residues. Here we identify an exosite on the SOCS2-SH2 domain which, when bound to a non-phosphorylated peptide (F3), enhances SH2 affinity for canonical phosphorylated ligands. Solution of the SOCS2/F3 crystal structure reveals F3 as an α-helix which binds on the opposite side of the SH2 domain to the phosphopeptide binding site. F3:exosite binding appears to stabilise the SOCS2-SH2 domain, resulting in slower dissociation of phosphorylated ligands and consequently, enhances binding affinity. This biophysical enhancement of SH2:pTyr binding affinity translates to increase SOCS2 inhibition of GH signaling

Identification and validation of FGFR2 mutations providing resistance to pan-FGFR inhibitor BGJ398

Endometrial cancer (EC) is the most commonly diagnosed gynaecological cancer, and is responsible for ~370 deaths per year in Australia and 8600 deaths annually in the USA. Fibroblast growth factor receptor 2 (FGFR2) mutations have been identified in ~ 12% of endometrial cancer patients and research confirms it is a valid therapeutic target. Tyrosine kinase inhibitors (TKIs) have been used in the last few years to treat patients with mutant receptor tyrosine kinases (RTKs). Despite patients showing an initial response to these TKIs, acquired resistance associated with cancer relapse often occurs. Acquired resistance is frequently caused by secondary mutations in the kinase domain that either directly hinder drug binding or stabilise a conformation not conducive to drug binding. In recent years, the Ba/F3 cell line model system has been used to identify mutations causing resistance to these inhibitors and many of these mutations have subsequently been identified in patients treated with these TKIs. We sought to identify FGFR2 kinase domain mutations that confer resistance to the pan-FGFR inhibitor BGJ398. We cultured Ba/F3 cells expressing FGFR2 in high doses of BGJ398 and identified 6 resistant clones harbouring either the FGFR2E566A or FGFR2V565I mutations in the kinase domain. Ba/F3 cells carrying each mutations, together with the FGFR2N550K mutation commonly seen in patients, were used to assess if these mutations were cross-resistant to other FGFR inhibitors (PD173074, ponatinib, AZD4547 and LY2874455). Only LY2874455 inhibited all the resistant FGFR2 mutations. In addition, lentiviral transduction of the endometrial cancer cell line JHUEM2 with the same FGFR2 resistance mutations resulted in heterozygous expression of the resistance alleles confirmed by sequencing cDNA. Transduced JHUEM2 cell lines were tested against the panel of FGFR inhibitors, however, the same level of resistance that was seen in Ba/F3 cells was not always observed in the JHUEM2 cell lines. We also used the myeloma cell line KMS11-R, which harbours a heterozygous FGFR3V565I mutation, and showed these cells conferred resistance to all inhibitors except LY2874455. From these data we propose that tumours harbouring FGFR mutations should be treated with LY2874455, as it effectively inhibits all identified FGFR mutations that cause resistance to other FGFR inhibitors

Application of the Matrix Pencil Method to Rational Fitting of Frequency-Domain Responses

This paper presents a general methodology based on the matrix pencil method (MPM) for the fitting of frequency-domain responses in order to be properly represented in time-domain analysis. By virtue of the proposed method, a rational fitting of the frequency-domain responses can be inferred, which, in turn, helps with their inclusion into time-domain calculations. The proposed technique is well suited for the estimation of any type of function including the case of those with superimposed noise. The main feature of the method is its direct solution, hence avoiding any iteration in the estimation process. Moreover, the method does not require starting poles as opposed to the vector-fitting (VF) method. This paper presents the validation of the proposed approach by fitting the frequency responses of general a priori known functions, artificially created noisy functions and those of power components, namely: the elements of a power transformer admittance matrix and the characteristic admittance and propagation functions of a single-core sheathed cable. Finally, a time-domain analysis referring to lightning transients on a transmission line is presented

The growth hormone receptor: mechanism of receptor activation, cell signaling, and physiological aspects

The growth hormone receptor (GHR), although most well known for regulating growth, has many other important biological functions including regulating metabolism and controlling physiological processes related to the hepatobiliary, cardiovascular, renal, gastrointestinal, and reproductive systems. In addition, growth hormone signaling is an important regulator of aging and plays a significant role in cancer development. Growth hormone activates the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, and recent studies have provided a new understanding of the mechanism of JAK2 activation by growth hormone binding to its receptor. JAK2 activation is required for growth hormone-mediated activation of STAT1, STAT3, and STAT5, and the negative regulation of JAK-STAT signaling comprises an important step in the control of this signaling pathway. The GHR also activates the Src family kinase signaling pathway independent of JAK2. This review covers the molecular mechanisms of GHR activation and signal transduction as well as the physiological consequences of growth hormone signaling

The N550K/H mutations in FGFR2 confer differential resistance to PD173074, dovitinib and ponatinib ATP-competitive inhibitors

We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified fourteen dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2mutant endometrial cancers (EC). Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors. Whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2mutant cell lines, 3/7 showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2N550K in JHUEM-2 cells (FGFR2C383R) conferred resistance (~5 fold) to PD173074, providing independent data that FGFR2N550K can be associated with drug resistance. Biochemical in vitro kinase analyses also shows ponatinib is more effective than dovitinib at inhibiting FGFR2N550K. We propose tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase

Tyrosine kinases compete for growth hormone receptor binding and regulate receptor mobility and degradation

Summary: Growth hormone (GH) acts via JAK2 and LYN to regulate growth, metabolism, and neural function. However, the relationship between these tyrosine kinases remains enigmatic. Through an interdisciplinary approach combining cell biology, structural biology, computation, and single-particle tracking on live cells, we find overlapping LYN and JAK2 Box1-Box2-binding regions in GH receptor (GHR). Our data implicate direct competition between JAK2 and LYN for GHR binding and imply divergent signaling profiles. We show that GHR exhibits distinct mobility states within the cell membrane and that activation of LYN by GH mediates GHR immobilization, thereby initiating its nanoclustering in the membrane. Importantly, we observe that LYN mediates cytokine receptor degradation, thereby controlling receptor turnover and activity, and this applies to related cytokine receptors. Our study offers insight into the molecular interactions of LYN with GHR and highlights important functions for LYN in regulating GHR nanoclustering, signaling, and degradation, traits broadly relevant to many cytokine receptors