Biology subjects

Goldsmith, E. J.

Publications and source records attributed to Goldsmith, E. J..

3 recordsLinked to original sources

bioRxiv2023

Water and chloride as allosteric inhibitors in WNK kinase osmosensing

Osmotic stress and chloride regulate the autophosphorylation and activity of the WNK1 and WNK3 kinase domains. The kinase domain of unphosphorylated WNK1 (uWNK1) is an asymmetric dimer possessing water molecules conserved in multiple uWNK1 crystal structures. Conserved waters are present in two networks, referred to here as conserved water networks 1 and 2 (CWN1and CWN2). Here we show that PEG400 applied to crystals of dimeric uWNK1 induces de-dimerization. Both the WNK1 the water networks and the chloride binding site and are disrupted by PEG400. CWN1 is surrounded by a cluster of pan-WNK-conserved charged residues. Here we mutagenized these charges in WNK3, a highly active WNK isoform kinase domain, and WNK1, the isoform best studied crystallographically. Mutation of E314 in the Activation Loop of WNK3 (WNK3/E314Q and WNK3/E314A, and the homologous WNK1/E388A) enhanced the rate of autophosphorylation, and reduced chloride sensitivity. Other WNK3 Cluster mutants reduced the rate of autophosphorylation activity coupled with greater chloride sensitivity than wild-type. The water and chloride regulation thus appear linked. The lower activity of some mutants may reflect effects on catalysis. Crystallography showed that activating mutants introduced conformational changes in similar parts of the structure to those induced by PEG400. WNK activating mutations and crystallography support a role for CWN1 in WNK inhibition consistent with water functioning as an allosteric ligand.

biophysics↗

bioRxiv2021

WNK1 Enhances Migration and Invasion in Breast Cancer Models

Metastasis is the major cause of mortality in breast cancer patients. Many signaling pathways have been linked to cancer invasiveness, but blockade of few protein components has succeeded in reducing metastasis. Thus, identification of proteins contributing to invasion that are manipulable by small molecules may be valuable in inhibiting spread of the disease. The protein kinase WNK1 (with no lysine (K) 1) has been suggested to induce migration of cells representing a range of cancer types. Analyses of mouse models and patient data have implicated WNK1 as one of a handful of genes uniquely linked to invasive breast cancer. Here we present evidence that inhibition of WNK1 slows breast cancer metastasis. We show that depletion or inhibition of WNK1 reduces migration of several breast cancer cell lines in wound healing assays and decreases invasion in collagen matrices. Furthermore, WNK1 depletion suppresses expression of AXL, a tyrosine kinase implicated in metastasis. Finally, we demonstrate that WNK inhibition in mice attenuates tumor progression and metastatic burden. These data showing reduced migration, invasion, and metastasis upon WNK1 depletion in multiple breast cancer models suggest that WNK1 contributes to the metastatic phenotype and that WNK1 inhibition may offer a therapeutic avenue for attenuating progression of invasive breast cancers.

cancer biology↗

bioRxiv2018

Anatomy of a pressure sensing protein kinase

Cells respond to hydrostatic pressure to maintain cellular, organ, and organism level functions, yet the direct pressure sensors are largely unknown. Here we show that hydrostatic pressure directly activates With No Lysine(K) kinase-3 (WNK3) 1, a soluble intracellular protein kinase. Using gel filtration we demonstrate that pressure induces a dimer to monomer transition in a construct of the unphosphorylated kinase domain of WNK3 (uWNK3-KDm or uWNK3). The uWNK3 has not been crystallized, but crosslinking data suggest that the uWNK3 dimer corresponds to crystallographically observed dimer of WNK1 (uWNK1-KDm, or uWNK1) 2,3. Sequence alignments with WNKs from species living in different pressure environments and mutational analysis lend further support for this idea. Unique features of the uWNK1 structure suggest a mechanism involving bound water. We further show that hydrostatic pressure activates full-length WNK3 in D. melanogaster tubules.

biophysics↗