Rabbit Anti-Human WNK1 (phospho Thr60) (a.a. 24-73) Polyclonal antibody

The nutrient-responsive beta-O-linked N-acetylglucosamine (O-GlcNAc) modification of critical effector proteins modulates signaling and transcriptional pathways contributing to cellular development and survival. An elevation in global protein O-GlcNAc modification occurs during the early stages of osteoblast differentiation and correlates with enhanced transcriptional activity of RUNX2, a key regulator of osteogenesis. To identify other substrates of O-GlcNAc transferase in differentiating MC3T3E1 osteoblasts, O-GlcNAc-modified peptides were enriched by wheat germ agglutinin lectin weak affinity chromatography and identified by tandem mass spectrometry using electron transfer dissociation. This peptide fragmentation approach leaves the labile O-linkage intact permitting direct identification of O-GlcNAc-modified peptides. O-GlcNAc modification was observed on enzymes involved in post-translational regulation, including MAST4 and WNK1 kinases, a ubiquitin-associated protein (UBAP2l), and the histone acetyltransferase CREB-binding protein. CREB-binding protein, a transcriptional co-activator that associates with CREB and RUNX2, is O-GlcNAcylated at Ser-147 and Ser-2360, the latter of which is a known site of phosphorylation. Additionally, O-GlcNAcylation of components of the TGF beta-activated kinase 1 (TAK1) signaling complex, TAB1 and TAB2, occurred in close proximity to known sites of Ser/Thr phosphorylation and a putative nuclear localization sequence within TAB2. These findings demonstrate the presence of O-GlcNAc modification on proteins critical to bone formation, remodeling, and fracture healing and will enable evaluation of this modification on protein function and regulation. Molecular & Cellular Proteomics 12: 10.1074/mcp.M112.026633, 945-955, 2013.

Polymorphisms in the gene encoding sterile 20/SPS1-related proline/alanine-rich kinase (SPAK) associate with hypertension susceptibility in humans SPAK interacts with WNK kinases to regulate the Na+-K+ 2Cl(-) and Na+-Cl- co-transporters [collectively, N(K)CC] Mutations in WNK1/4 and N(K)CC can cause changes in BP and dyskalemia in humans but the physiologic role of SPAK in vivo is unknown We generated and analyzed SPAK null mice by targeting disruption of exons 9 and 10 of SPAK Compared with SPAK(+/+) littermates, SPAK(+/-) mice exhibited hypotension without significant electrolyte abnormalities, and SPAK(-/-) mice not only exhibited hypotension but also recapitulated Gitelman syndrome with hypokalemia hypomagnesemia, and hypocalciuria In the kidney tissues of SPAK(-/-) mice the expression of total and phosphorylated (p)NCC was markedly decreased, but that of p-OSR1 total NKCC2, and p NKCC2 was significantly increased We observed a blunted response to thiazide but normal response to furosemide in SPAK(-/-) mice In aortic tissues total NKCC1 expression was increased but p-NKCC1 was decreased in SPAK-deficient mice Both SPAK(+/-) and SPAK(-/-) mice had impaired responses to the selective alpha(1)-adrenergic agonist phenylephrine and the NKCC1 inhibitor bumetanide suggesting that impaired aortic contractility may contribute to the hypotension of SPAK null mice In summary, SPAK-null mice have defects of NCC in the kidneys and NKCC1 in the blood vessels, leading to hypotension through renal salt wasting and vasodilation SPAK may be a promising target for antihypertensive therapy