Structural basis of the regulatory mechanism of the plant CIPK family of protein kinases controlling ion homeostasis and abiotic stress

Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calcium-dependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases.Cell perception of extracellular stimuli is followed by a transient variation in cytosolic calcium concentration. Plants have evolved to produce the specific molecular machinery to interpret this primary information and to transmit this signal to the components that organize the cell response (1–4). The plant family of serine/threonine protein kinases PKS or CIPKs (hereinafter CIPKs) and their activators, the calcium-binding proteins SCaBPs or CBLs (hereinafter CBLs) (5, 6) function together in decoding calcium signals caused by different environmental stimuli. Available data suggest a mechanism in which calcium mediates the formation of stable CIPK–CBL complexes that regulate the phosphorylation state and activity of various ion transporters involved in the maintenance of cell ion homeostasis and abiotic stress responses in plants. Among them, the Arabidopsis thaliana CIPK24/SOS2-CBL4/SOS3 complex activates the Na⁺/H⁺ antiporter SOS1 to maintain intracellular levels of the toxic Na⁺ low under salt stress (7–9), the CIPK11–CBL2 pair regulates the plasma membrane H⁺-ATPase AHA2 to control the transmembrane pH gradient (10), the CIPK23–CBL1/9 (11, 12) regulates the activity of the K⁺ transporter AKT1 to increase the plant K⁺ uptake capability under limiting K⁺ supply conditions (12, 13), and CIPK23–CBL1 mediates nitrate sensing and uptake by phosphorylation of the nitrate transporter CHL1 (14). Together these findings show that understanding the molecular mechanisms underling CIPKs function provides opportunities to increase plant tolerance to abiotic stress and to improve plants for human benefit.CIPKs and CBLs contain discrete structural modules that are involved in the calcium-dependent regulation of the activity of the system and ensure the colocalization of the CIPK–CBL interacting pairs with their substrates at particular sites within the cell (15–17). CIPKs include an N-terminal kinase catalytic domain followed by a characteristic self-inhibitory motif known as FISL or NAF motif (hereinafter NAF, Pfam no. PF03822) (1, 6) and a protein phosphatase 2C binding domain designated as PPI (11, 18, 19). The NAF motif directly interacts with the catalytic domain and inhibits the kinase activity. The calcium-dependent interaction of CBLs with the NAF motif relieves the self-inhibition and activates the CIPKs (5, 6, 19, 20). The calcium binding to CBLs is mediated by four EF hand-like calcium binding motives. In addition, several CBLs are myristoylated and/or palmitoylated. These modifications are essential for recruiting their interacting CIPK partner to the plasma or vacuolar membrane (17, 21–23), and they may also be involved in the interaction of the CIPK–CBL complexes with their substrates (24). In addition, the phosphorylation of a conserved serine residue at the C terminus of CBLs by its interacting CIPK is required for activation of transporter substrates. It has been proposed that this process may stabilize the CIPK–CBL complex and trigger conformational changes to the binary complex that enhance its specificity toward target proteins (13, 25).Like many other kinases, CIPKs are also regulated by the phosphorylation of the activation loop by upstream kinases. This loop undergoes large conformational changes upon phosphorylation, allowing the entrance and the stabilization of substrates at the kinase active site (26). The activation loop of the CIPKs contains three conserved Tyr, Thr, or Ser residues. For some members of the family, the mutation of one of these residues to Asp mimics phosphorylation and produces the activation of the kinase, partly overcoming the effect of the self-inhibitory NAF motif. In fact, these phosphorylation-mimicking mutations and the deletion of the inhibitory domain produce a synergistic effect on the CIPK activity (6, 27–29). Transgenic plants expressing these CIPK24/SOS2 mutant proteins show improved salt tolerance (30).The kinase self-phosphorylation is another regulatory mechanism used by CIPKs. CIPK24/SOS2 is able to self-phosphorylate, and the autophosphorylation is important for its activity (31). Although the default state of CIPKs is inactive, some degree of autophosphorylation activity has been observed even for dephosphorylated and CBL-unbound CIPKs, which suggests that some CIPKs display basal activity (6). Indeed, it has been shown that the general regulatory factor 14-3-3 proteins (32) interact with CIPK24/SOS2 and repress its basal kinase activity when plants are grown in the absence of salt stress (33).The crystal structure of the binary complex of Ca²⁺-CBL4/SOS3 with the C-terminal regulatory moiety of CIPK24/SOS2 revealed the molecular mechanism underlying CBL-mediated activation of the CIPKs. The structure showed that the CIPK24/SOS2 self-inhibitory NAF motif is bound to CBL4/SOS3 and, consequently, it is not accessible to the kinase domain (19, 20). However, whether the CBL-unbound NAF blocks the active site or inhibits the enzyme by an allosteric mechanism is not known. To determine the molecular and structural basis for the CIPKs autoinhibition by the NAF and the activation by upstream kinases, we solved the structures of CIPK23 and CIPK24/SOS2. Our data show that inactivation of the kinases relies on the blockage of the active site by the NAF motif and the activation loop, which constitutes the basis for the conserved NAF-mediated self-inhibition of the CIPKs.     

Outcomes of preoperative Angiotensin-converting enzyme inhibitor therapy in patients undergoing isolated coronary artery bypass grafting

The association between preoperative use of angiotensin-converting enzyme (ACE) inhibitors and outcomes after coronary artery bypass grafting (CABG) remain controversial. Our aim was to study in-hospital outcomes after isolated CABG in patients on preoperative ACE inhibitors. A retrospective analysis of 8,889 patients who underwent isolated CABG from 2000 through 2011 was conducted. The primary outcome of interest was the incidence of major adverse events (MAEs) defined as a composite of mortality, postoperative renal dysfunction, myocardial infarction, stroke, and atrial fibrillation during index hospitalization. The secondary outcome was the incidence of individual outcomes included in MAEs. Logistic regression analyses were performed. Of 8,889 patients, 3,983 (45%) were on preoperative ACE inhibitors and 4,906 (55%) were not. Overall incidence of MAEs was 38.1% (n = 1,518) in the ACE inhibitor group compared to 33.6% (n = 1,649) in the no-ACE inhibitor group. Preoperative use of ACE inhibitors was independently associated with MAEs (odds ratio 1.13, 95% confidence interval 1.03 to 1.24), most of which was driven by a statistically significant increase in postoperative renal dysfunction (odds ratio 1.18, 95% confidence interval 1.03 to 1.36) and atrial fibrillation (odds ratio 1.15, 95% confidence interval 1.05 to 1.27). In-hospital mortality, postoperative myocardial infarction, and stroke were not significantly associated with preoperative ACE inhibitor use. Analyses performed after excluding patients with low ejection fractions yielded similar results. In conclusion, preoperative ACE inhibitor use was associated with an increased risk of MAEs after CABG, in particular postoperative renal dysfunction and atrial fibrillation.     

Characterizing Changes in the Excitability of Corticospinal Projections to Proximal Muscles of the Upper Limb

BackgroundThere has been an explosion of interest in methods of exogenous brain stimulation that induce changes in the excitability of human cerebral cortex. The expectation is that these methods may promote recovery of function following brain injury. To assess their effects on motor output, it is typical to assess the state of corticospinal projections from primary motor cortex to muscles of the hand, via electromyographic responses to transcranial magnetic stimulation. If a range of stimulation intensities is employed, the recruitment curves (RCs) obtained can, at least for intrinsic hand muscles, be fitted by a sigmoid function.Objective/hypothesisTo establish whether sigmoid fits provide a reliable basis upon which to characterize the input–output properties of the corticospinal pathway for muscles proximal to the hand, and to assess as an alternative the area under the (recruitment) curve (AURC).MethodsA comparison of the reliability of these measures, using RCs obtained for muscles that are frequently the targets of rehabilitation.ResultsThe AURC is an extremely reliable measure of the state of corticospinal projections to hand and forearm muscles, which has both face and concurrent validity. Construct validity is demonstrated by detection of widely distributed (across muscles) changes in corticospinal excitability induced by paired associative stimulation (PAS).Conclusion(s)The parameters derived from sigmoid fits are unlikely to provide an adequate means to assess the effectiveness of therapeutic regimes. The AURC can be employed to characterize corticospinal projections to a range of muscles, and gauge the efficacy of longitudinal interventions in clinical rehabilitation.     

Impact of incident comorbidity on functional loss in elderly chronic kidney disease patients undergoing hemodialysis

The incidence of end stage renal disease in older persons has been increasing progressively over the last 10 years. Improved survival rates with renal replacement therapy are making this increased prevalence even more pronounced. The usual risks of morbidity and requirements for specialized care associated with older people increase dramatically when they have chronic kidney disease (CKD). It has been seen that the majority of patients in hemodialysis units are over the age of 60, and have significant co-morbidities. The relationship between older age, chronic disorders and functional dependence (FD) is well known. Accordingly, nursing care planning must be designed with this in mind. The aim of this study was to assess whether the comorbidity associated with CKD modifies FD in patients on hemodialysis. We undertook a prospective longitudinal cohort study of hemodialysis outpatients in Málaga, Spain, using the Barthel test to establish FD and the Charlson comorbidity index to quantify comorbidity. All health events were analyzed to select those study patients with incident comorbidity, understood as the appearance of a new disease that could modify the Charlson comorbidity index, and determine the change in FD. Multivariate linear regression showed that the best model for predicting functional loss was that which considered comorbidity adjusted for age, particularly when it occurred as a result of hospital admission, as it was shown to have an important predictive value for the onset of a decrease in functional dependency scores in patients with CKD.     

Sodium Channel Blockers Modulate Abnormal Activity of Regenerating Nociceptive Corneal Nerves After Surgical Lesion

PurposeTo test the effect of different sodium channel blockers on the electrical activity of corneal nociceptors in intact and surgically injured corneas.MethodsIn anesthetized guinea pigs, a 4-mm diameter corneal flap was performed in one eye at a midstromal depth using a custom-made microkeratome. At different times after surgery (3 hours to 15 days), the electrical activity of corneal nociceptor fibers was recorded from ciliary nerve filaments in the superfused eye in vitro. Mechanical threshold was measured using calibrated von Frey hairs; chemical stimulation was performed applying 30-second CO₂ gas pulses. The characteristics of the spontaneous and stimulus-evoked activity of corneal nociceptors recorded from intact and lesioned corneas, before and after treatment with the sodium channel blockers lidocaine, carbamazepine, and amitriptyline, were compared.ResultsNo spontaneous or stimulus-evoked impulse activity was detected inside the flap at any of the studied time points. However, both were recorded from mechanonociceptor and polymodal nociceptors fibers in the surrounding corneal tissue, being significantly higher (sensitization) 24 to 48 hours after surgery. In these fibers, none of the tested drugs affected mechanical threshold, but they significantly reduced the CO₂ response of polymodal nociceptors of intact and injured corneas. Likewise, they diminished significantly the transient increase in spontaneous and stimulus-evoked activity of sensitized polymodal nociceptors.ConclusionsNa⁺ channel blockers decrease the excitability of intact and sensitized corneal nociceptor fibers, thus acting as potential tools to attenuate their abnormal activity, which underlies the spontaneous pain, hyperalgesia, and allodynia often accompanying surgical corneal lesions, as occurs after photorefractive surgery.