Activity of heterologously expressed NKCC1 was analyzed under basal and activated conditions in the presence and absence of binding of Ste20-related prolinealanine-rich kinase (SPAK). Mutant NKCC1 that lacks the ability to bind to this kinase showed K ؉ transport function identical to wild-type NKCC1. Thus, preventing the binding of the kinase to the cotransporter does not affect cotransporter function. In contrast, several experiments suggest a possible role for SPAK as a scaffolding protein. First, Western blot analysis revealed the presence, and in some tissues abundance, of truncated forms of SPAK and OSR1 in which the kinase domains are affected and thus lack kinase activity. Second, a yeast two-hybrid screen of proteins that interact with the regulatory (binding) domain of SPAK identified several proteins all involved in cellular stress pathways. Third, p38, one of the three major MAPKs, can be coimmunoprecipitated with SPAK and with NKCC1 in an activitydependent manner. The amount of p38 coimmunoprecipitated with the kinase and the cotransporter significantly decreases upon cellular stress, whereas the interaction of the kinase with NKCC1 remains unchanged. These findings suggest that cation-chloride cotransporters might act as "sensors" for cellular stress, and SPAK, by interacting with the cotransporter, serves as an intermediate in the response to cellular stress. Cation-chloride cotransporters, which mediate the tightly coupled, electroneutral movement of cations (Na ϩ and K ϩ ) together with Cl Ϫ , can be divided into Na ϩ -dependent transporters, such as Na-K-2Cl cotransporters (NKCC1-2) and the Na-Cl cotransporter (NCC), and Na ϩ -independent K-Cl cotransporters (KCC1-4). All of these transporters have a well conserved topology with large intracellular amino-terminal and carboxyl-terminal tails and 12 transmembrane spanning domains. The core protein shares some homology to amino acid permeases (for reviews see Refs. 1 and 2). A variety of stimuli regulates these transporters, including hormonal (3), cytokines (4, 5), cell volume (6, 7), oxidative stress (8), etc. There is also accumulating evidence that cation-chloride cotransporters participate in pathways leading to cell differentiation, growth and proliferation (9, 10), and apoptosis (11,12). At the molecular level, the activation-deactivation of these cotransporters mostly involves phosphorylation/dephosphorylation mechanisms, the details of which are still the subject of intense investigation.To identify proteins that directly interact and regulate cation-chloride cotransporters, we recently performed a yeast twohybrid screen using the cytosolic amino-terminal tail of KCC3. We identified two closely related kinases that bind to KCC3, NKCC1 and NKCC2 (13). These kinases belong to the group of Ste20 kinases that function as regulators of MAPK 1 cascades (14). The first kinase, SPAK (Ste-20 related proline-alaninerich kinase, or PASK, as the rat homologue), is highly expressed in epithelia and neurons (15). Its gene is located on human chromosome...
GABAergic and glycinergic function is dependent on neuronal intracellular chloride. The neuron-specific electroneutral potassium (K+) and chloride (Cl-) cotransporter (KCC2), is a key regulator of neuronal Cl-, yet little is known about KCC2 regulation. Using yeast two-hybrid, we identified Protein Associated with Myc (PAM) as a binding partner of KCC2. The RCC1 (Regulator of Chromatin Condensation) domain of PAM binds to the carboxyl terminus of KCC2, as demonstrated through yeast two-hybrid and GST-pull-down assays. RCC1/PAM and full-length KCC2 coimmunoprecipitate following heterologous co-expression in HEK293 cells. Additionally, 86Rb/K+ uptake assays in this model system show that RCC1/PAM causes increased KCC2-mediated flux. After narrowing down RCC1/PAM binding to a 20 amino acid region on the KCC2 carboxyl terminus, we created a point mutant in this region to eliminate interaction between the KCC2 carboxyl terminus and RCC1/PAM. This same mutation abolishes N-ethylmaleimide activation of KCC2, suggesting that PAM plays a role in modulating KCC2 function.
Despite the political, ethical and financial complexities, there are some human diseases that researchers feel can only be understood by working with non-human primates. Nicole Garbarini investigates.
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