OSR1 and SPAK cooperatively modulate Sertoli cell support of mouse spermatogenesis.
ABSTRACT: We investigated the role of oxidative stress-responsive kinase-1 (OSR1) and STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase (SPAK), upstream regulators of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1)-essential for spermatogenesis-in mouse models of male fertility. Global OSR1(+/-) gene mutations, but not global SPAK(-/-) or Sertoli cell (SC)-specific OSR1 gene knockout (SC-OSR1(-/-)), cause subfertility with impaired sperm function and are associated with reduced abundance of phosphorylated (p)-NKCC1 but increased p-SPAK expression in testicular tissue and spermatozoa. To dissect further in a SC-specific manner the compensatory effect of OSR1 and SPAK in male fertility, we generated SC-OSR1(-/-) and SPAK(-/-) double knockout (DKO) male mice. These are infertile with defective spermatogenesis, presenting a SC-only-like syndrome. Disrupted meiotic progression and increased germ cell apoptosis occurred in the first wave of spermatogenesis. The abundance of total and p-NKCC1 was significantly decreased in the testicular tissues of DKO mice. These results indicate that OSR1 and SPAK cooperatively regulate NKCC1-dependent spermatogenesis in a SC-restricted manner.
Project description:The SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1) kinases interact and phosphorylate NKCC1 (Na+-K+-2Cl- co-transporter-1), leading to its activation. Recent studies indicated that SPAK and OSR1 are phosphorylated and activated by the WNK1 [with no K (lysine) protein kinase-1] and WNK4, genes mutated in humans affected by Gordon's hypertension syndrome. In the present study, we have identified three residues in NKCC1 (Thr175/Thr179/Thr184 in shark or Thr203/Thr207/Thr212 in human) that are phosphorylated by SPAK and OSR1, and have developed a peptide substrate, CATCHtide (cation chloride co-transporter peptide substrate), to assess SPAK and OSR1 activity. Exposure of HEK-293 (human embryonic kidney) cells to osmotic stress, which leads to phosphorylation and activation of NKCC1, increased phosphorylation of NKCC1 at the sites targeted by SPAK/OSR1. The residues on NKCC1, phosphorylated by SPAK/OSR1, are conserved in other cation co-transporters, such as the Na+-Cl- co-transporter, the target of thiazide drugs that lower blood pressure in humans with Gordon's syndrome. Furthermore, we characterize the properties of a 92-residue CCT (conserved C-terminal) domain on SPAK and OSR1 that interacts with an RFXV (Arg-Phe-Xaa-Val) motif present in the substrate NKCC1 and its activators WNK1/WNK4. A peptide containing the RFXV motif interacts with nanomolar affinity with the CCT domains of SPAK/OSR1 and can be utilized to affinity-purify SPAK and OSR1 from cell extracts. Mutation of the arginine, phenylalanine or valine residue within this peptide abolishes binding to SPAK/OSR1. We have identified specific residues within the CCT domain that are required for interaction with the RFXV motif and have demonstrated that mutation of these in OSR1 inhibited phosphorylation of NKCC1, but not of CATCHtide which does not possess an RFXV motif. We establish that an intact CCT domain is required for WNK1 to efficiently phosphorylate and activate OSR1. These data establish that the CCT domain functions as a multipurpose docking site, enabling SPAK/OSR1 to interact with substrates (NKCC1) and activators (WNK1/WNK4).
Project description:In immature neurons the amino acid neurotransmitter, GABA provides the dominant mode for neuronal excitation by inducing membrane depolarization due to Cl(-) efflux through GABA(A) receptors (GABA(A)Rs). The driving force for Cl(-) is outward because the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) elevates the Cl(-) concentration in these cells. GABA-induced membrane depolarization and the resulting activation of voltage-gated Ca(2+) channels is fundamental to normal brain development, yet the mechanisms that regulate depolarizing GABA are not well understood. The neurosteroid estradiol potently augments depolarizing GABA action in the immature hypothalamus by enhancing the activity of the NKCC1 cotransporter. Understanding how estradiol controls NKCC1 activity will be essential for a complete understanding of brain development. We now report that estradiol treatment of newborn rat pups significantly increases protein levels of two kinases upstream of the NKCC1 cotransporter, SPAK (STE20/SPS1-related proline alanine rich kinase) and OSR1 (oxidative stress response kinase). The estradiol-induced increase is transcription dependent, and its time course parallels that of estradiol-enhanced phosphorylation of NKCC1. Antisense oligonucleotide-mediated knockdown of SPAK, and to a lesser degree of OSR1, precludes estradiol-mediated enhancement of NKCC1 phosphorylation. Functionally, knockdown of SPAK or OSR1 in embryonic hypothalamic cultures diminishes estradiol-enhanced Ca(2+) influx induced by GABA(A)R activation. Our data suggest that SPAK and OSR1 may be critical factors in the regulation of depolarizing GABA-mediated processes in the developing brain. It will be important to examine these kinases with respect to sex differences and developmental brain anomalies in future studies.
Project description:Wnk kinase maintains cell volume, regulating various transporters such as sodium-chloride cotransporter, potassium-chloride cotransporter, and sodium-potassium-chloride cotransporter 1 (NKCC1) through the phosphorylation of oxidative stress responsive kinase 1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). However, the activating mechanism of Wnk kinase in specific tissues and specific conditions is broadly unclear. In the present study, we used a human salivary gland (HSG) cell line as a model and showed that Ca(2+) may have a role in regulating Wnk kinase in the HSG cell line. Through this study, we found that the HSG cell line expressed molecules participating in the WNK-OSR1-NKCC pathway, such as Wnk1, Wnk4, OSR1, SPAK, and NKCC1. The HSG cell line showed an intracellular Ca(2+) concentration ([Ca(2+)]i) increase in response to hypotonic stimulation, and the response was synchronized with the phosphorylation of OSR1. Interestingly, when we inhibited the hypotonically induced [Ca(2+)]i increase with nonspecific Ca(2+) channel blockers such as 2-aminoethoxydiphenyl borate, gadolinium, and lanthanum, the phosphorylated OSR1 level was also diminished. Moreover, a cyclopiazonic acid-induced passive [Ca(2+)]i elevation was evoked by the phosphorylation of OSR1, and the amount of phosphorylated OSR1 decreased when the cells were treated with BAPTA, a Ca(2+) chelator. Finally, through that process, NKCC1 activity also decreased to maintain the cell volume in the HSG cell line. These results indicate that Ca(2+) may regulate the WNK-OSR1 pathway and NKCC1 activity in the HSG cell line. This is the first demonstration that indicates upstream Ca(2+) regulation of the WNK-OSR1 pathway in intact cells.
Project description:Mouse protein-25 (MO25) isoforms bind to the STRAD pseudokinase and stabilise it in a conformation that can activate the LKB1 tumour suppressor kinase. We demonstrate that by binding to several STE20 family kinases, MO25 has roles beyond controlling LKB1. These new MO25 targets are SPAK/OSR1 kinases, regulators of ion homeostasis and blood pressure, and MST3/MST4/YSK1, involved in controlling development and morphogenesis. Our analyses suggest that MO25? and MO25? associate with these STE20 kinases in a similar manner to STRAD. MO25 isoforms induce approximately 100-fold activation of SPAK/OSR1 dramatically enhancing their ability to phosphorylate the ion cotransporters NKCC1, NKCC2 and NCC, leading to the identification of several new phosphorylation sites. siRNA-mediated reduction of expression of MO25 isoforms in mammalian cells inhibited phosphorylation of endogenous NKCC1 at residues phosphorylated by SPAK/OSR1, which is rescued by re-expression of MO25?. MO25?/? binding to MST3/MST4/YSK1 also stimulated kinase activity three- to four-fold. MO25 has evolved as a key regulator of a group of STE20 kinases and may represent an ancestral mechanism of regulating conformation of pseudokinases and activating catalytically competent protein kinases.
Project description:The pivotal role of KCC2 and NKCC1 in development and maintenance of fast inhibitory neurotransmission and their implication in severe human diseases arouse interest in posttranscriptional regulatory mechanisms such as (de)phosphorylation. Staurosporine (broad kinase inhibitor) and N-ethylmalemide (NEM) that modulate kinase and phosphatase activities enhance KCC2 and decrease NKCC1 activity. Here, we investigated the regulatory mechanism for this reciprocal regulation by mass spectrometry and immunoblot analyses using phospho-specific antibodies. Our analyses revealed that application of staurosporine or NEM dephosphorylates Thr1007 of KCC2, and Thr203, Thr207 and Thr212 of NKCC1. Dephosphorylation of Thr1007 of KCC2, and Thr207 and Thr212 of NKCC1 were previously demonstrated to activate KCC2 and to inactivate NKCC1. In addition, application of the two agents resulted in dephosphorylation of the T-loop and S-loop phosphorylation sites Thr233 and Ser373 of SPAK, a critical kinase in the WNK-SPAK/OSR1 signaling module mediating phosphorylation of KCC2 and NKCC1. Taken together, these results suggest that reciprocal regulation of KCC2 and NKCC1 via staurosporine and NEM is based on WNK-SPAK/OSR1 signaling. The key regulatory phospho-site Ser940 of KCC2 is not critically involved in the enhanced activation of KCC2 upon staurosporine and NEM treatment, as both agents have opposite effects on its phosphorylation status. Finally, NEM acts in a tissue-specific manner on Ser940, as shown by comparative analysis in HEK293 cells and immature cultured hippocampal neurons. In summary, our analyses identified phospho-sites that are responsive to staurosporine or NEM application. This provides important information towards a better understanding of the cooperative interactions of different phospho-sites.
Project description:The oxidative-stress-responsive kinase 1 (OSR1) and the STE20/SPS1-related proline/alanine-rich kinase (SPAK) are key enzymes in a signalling cascade regulating the activity of Na(+)/K(+)/2Cl(-) co-transporters (NKCCs) in response to osmotic stress. Both kinases have a conserved carboxy-terminal (CCT) domain, which recognizes a unique peptide (Arg-Phe-Xaa-Val) motif present in OSR1- and SPAK-activating kinases (with-no-lysine kinase 1 (WNK1) and WNK4) as well as its substrates (NKCC1 and NKCC2). Here, we describe the structural basis of this recognition event as shown by the crystal structure of the CCT domain of OSR1 in complex with a peptide containing this motif, derived from WNK4. The CCT domain forms a novel protein fold that interacts with the Arg-Phe-Xaa-Val motif through a surface-exposed groove. An intricate web of interactions is observed between the CCT domain and an Arg-Phe-Xaa-Val motif-containing peptide derived from WNK4. Mutational analysis shows that these interactions are required for the CCT domain to bind to WNK1 and NKCC1. The CCT domain structure also shows how phosphorylation of a Ser/Thr residue preceding the Arg-Phe-Xaa-Val motif results in a steric clash, promoting its dissociation from the CCT domain. These results provide the first molecular insight into the mechanism by which the SPAK and OSR1 kinases specifically recognize their upstream activators and downstream substrates.
Project description:The oxidative-stress-responsive kinase 1 (OSR1) and the STE20/SPS1-related proline/alanine-rich kinase (SPAK) are key enzymes in a signaling cascade regulating the activity of Na(+)-K(+)-2Cl(-) cotransporters (NKCC1-2) and Na(+)-Cl(-) cotransporter (NCC). Both kinases have a conserved carboxyl-terminal (CCT) domain, which recognizes a unique peptide motif present in OSR1- and SPAK-activating kinases (with-no-lysine kinase 1 (WNK1) and WNK4) as well as their substrates (NKCC1, NKCC2, and NCC). Utilizing various modalities of the Rosetta Molecular Modeling Software Suite including flexible peptide docking and protein design, we comprehensively explored the sequence space recognized by the CCT domain. Specifically, we studied single residue mutations as well as complete unbiased designs of a hexapeptide substrate. The computational study started from a crystal structure of the CCT domain of OSR1 in complex with a hexapeptide derived from WNK4. Point mutations predicted to be favorable include Arg to His or Trp substitutions at position 2 and a Phe to Tyr substitution at position 3 of the hexapeptide. In addition, de novo design yielded two peptides predicted to bind to the CCT domain: FRFQVT and TRFDVT. These results, which indicate a little bit more freedom in the composition of the peptide, were confirmed through the use of yeast two-hybrid screening.
Project description:Na(+)-dependent chloride cotransporters (NKCC1, NKCC2, and NCC) are activated by phosphorylation to play critical roles in diverse physiological responses, including renal salt balance, hearing, epithelial fluid secretion, and volume regulation. Serine threonine kinase WNK4 (With No K = lysine member 4) and members of the Ste20 kinase family, namely SPAK and OSR1 (Ste20-related proline/alanine-rich kinase, Oxidative stress-responsive kinase) govern phosphorylation. According to present understanding, WNK4 phosphorylates key residues within SPAK/OSR1 leading to kinase activation, allowing SPAK/OSR1 to bind to and phosphorylate NKCC1, NKCC2, and NCC. Recently, the calcium-binding protein 39 (Cab39) has emerged as a binding partner and enhancer of SPAK/OSR1 activity, facilitating kinase autoactivation and promoting phosphorylation of the cotransporters. In the present study, we provide evidence showing that Cab39 differentially interacts with WNK4 and SPAK/OSR1 to switch the classic two kinase cascade into a signal kinase transduction mechanism. We found that WNK4 in association with Cab39 activates NKCC1 in a SPAK/OSR1-independent manner. We discovered that WNK4 possesses a domain that bears close resemblance to the SPAK/OSR1 C-terminal CCT/PF2 domain, which is required for physical interaction between the Ste20 kinases and the Na(+)-driven chloride cotransporters. Modeling, yeast two-hybrid, and functional data reveal that this PF2-like domain located downstream of the catalytic domain in WNK4 promotes the direct interaction between the kinase and NKCC1. We conclude that in addition to SPAK and OSR1, WNK4 is able to anchor itself to the N-terminal domain of NKCC1 and to promote cotransporter activation.
Project description:WNK kinases, including WNK3, and the associated downstream Ste20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress responsive 1 (OSR1) kinases, comprise an important signaling cascade that regulates the cation-chloride cotransporters. Ischemia-induced stimulation of the bumetanide-sensitive Na(+)-K(+)-Cl(-) cotransporter (NKCC1) plays an important role in the pathophysiology of experimental stroke, but the mechanism of its regulation in this context is unknown. Here, we investigated the WNK3-SPAK/OSR1 pathway as a regulator of NKCC1 stimulation and their collective role in ischemic brain damage.Wild-type WNK3 and WNK3 knockout mice were subjected to ischemic stroke via transient middle cerebral artery occlusion. Infarct volume, brain edema, blood brain barrier damage, white matter demyelination, and neurological deficits were assessed. Total and phosphorylated forms of WNK3 and SPAK/OSR1 were assayed by immunoblotting and immunostaining. In vitro ischemia studies in cultured neurons and immature oligodendrocytes were conducted using the oxygen-glucose deprivation/reoxygenation method.WNK3 knockout mice exhibited significantly decreased infarct volume and axonal demyelination, less cerebral edema, and accelerated neurobehavioral recovery compared with WNK3 wild-type mice subjected to middle cerebral artery occlusion. The neuroprotective phenotypes conferred by WNK3 knockout were associated with a decrease in stimulatory hyperphosphorylations of the SPAK/OSR1 catalytic T-loop and of NKCC1 stimulatory sites Thr(203)/Thr(207)/Thr(212), as well as with decreased cell surface expression of NKCC1. Genetic inhibition of WNK3 or small interfering RNA knockdown of SPAK/OSR1 increased the tolerance of cultured primary neurons and oligodendrocytes to in vitro ischemia.These data identify a novel role for the WNK3-SPAK/OSR1-NKCC1 signaling pathway in ischemic neuroglial injury and suggest the WNK3-SPAK/OSR1 kinase pathway as a therapeutic target for neuroprotection after ischemic stroke.
Project description:Na(+)-K(+)-2Cl(-) cotransporters (NKCCs), including NKCC1 and renal-specific NKCC2, and the Na(+)-Cl(-) cotransporter (NCC) play pivotal roles in the regulation of blood pressure (BP) and renal NaCl reabsorption. Oxidative stress-responsive kinase-1 (OSR1) is a known upstream regulator of N(K)CCs. We generated and analyzed global and kidney tubule-specific (KSP) OSR1 KO mice to elucidate the physiological role of OSR1 in vivo, particularly on BP and kidney function. Although global OSR1(-/-) mice were embryonically lethal, OSR1(+/-) mice had low BP associated with reduced phosphorylated (p) STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase (SPAK) and p-NKCC1 abundance in aortic tissue and attenuated p-NKCC2 abundance with increased total and p-NCC expression in the kidney. KSP-OSR1(-/-) mice had normal BP and hypercalciuria and maintained significant hypokalemia on a low-K(+) diet. KSP-OSR1(-/-) mice exhibited impaired Na(+) reabsorption in the thick ascending loop on a low-Na(+) diet accompanied by remarkably decreased expression of p-NKCC2 and a blunted response to furosemide, an NKCC2 inhibitor. The expression of total SPAK and p-SPAK was significantly increased in parallel to that of total NCC and p-NCC despite unchanged total NKCC2 expression. These results suggest that, globally, OSR1 is involved in the regulation of BP and renal tubular Na(+) reabsorption mainly via the activation of NKCC1 and NKCC2. In the kidneys, NKCC2 but not NCC is the main target of OSR1 and the reduced p-NKCC2 in KSP-OSR1(-/-) mice may lead to a Bartter-like syndrome.