Frequency-encoding Thr17 phospholamban phosphorylation is independent of Ser16 phosphorylation in cardiac myocytes

Both Ser(16) and Thr(17) of phospholamban (PLB) are phosphorylated, respectively, by cAMP-dependent protein kinase (PKA) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). PLB phosphorylation relieves cardiac sarcoplasmic reticulum Ca(2+) pump from inhibition by PLB. Previous studies have suggested that phosphorylation of Ser(16) by PKA is a prerequisite for Thr(17) phosphorylation by CaMKII and is essential to the relaxant effect of beta-adrenergic stimulation. To determine the role of Thr(17) PLB phosphorylation, we investigated the dual-site phosphorylation of PLB in isolated adult rat cardiac myocytes in response to beta(1)-adrenergic stimulation or electrical field stimulation (0. 1-3 Hz) or both. A beta(1)-adrenergic agonist, norepinephrine (10(-9)-10(-6) m), in the presence of an alpha(1)-adrenergic antagonist, prazosin (10(-6) m), selectively increases the PKA-dependent phosphorylation of PLB at Ser(16) in quiescent myocytes. In contrast, electrical pacing induces an opposite phosphorylation pattern, selectively enhancing the CaMKII-mediated Thr(17) PLB phosphorylation in a frequency-dependent manner. When combined, electric stimulation (2 Hz) and beta(1)-adrenergic stimulation lead to dual phosphorylation of PLB and exert a synergistic effect on phosphorylation of Thr(17) but not Ser(16). Frequency-dependent Thr(17) phosphorylation is closely correlated with a decrease in 50% relaxation time (t(50)) of cell contraction, which is independent of, but additive to, the relaxant effect of Ser(16) phosphorylation, resulting in hastened contractile relaxation at high stimulation frequencies. Thus, we conclude that in intact cardiac myocytes, phosphorylation of PLB at Thr(17) occurs in the absence of prior Ser(16) phosphorylation, and that frequencydependent Thr(17) PLB phosphorylation may provide an intrinsic mechanism for cardiac myocytes to adapt to a sudden change of heart rate.     

Phosphorylation-dependent conformational switch in spin-labeled phospholamban bound to SERCA

We have used chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) to probe the functional dynamics of phospholamban (PLB), which regulates the Ca-ATPase (SERCA) in cardiac sarcoplasmic reticulum. The transmembrane domain of PLB inhibits SERCA at low [Ca(2+)], but the cytoplasmic domain relieves this inhibition upon Ser16 phosphorylation. Monomeric PLB was synthesized with Ala11 replaced by the 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) spin label, which reports peptide backbone dynamics directly. PLB was reconstituted into membranes in the presence or absence of SERCA. TOAC-PLB showed normal inhibitory function, which was reversed by phosphorylation at Ser16 or by micromolar [Ca(2+)]. EPR showed that the PLB cytoplasmic domain exhibits two resolved conformations, a tense T state that is ordered and a relaxed R state that is dynamically disordered and extended. PLB phosphorylation shifts this equilibrium toward the R state and makes it more dynamic (hyperextended). Phosphorylation strongly perturbs the dynamics of SERCA-bound PLB without dissociating the complex, while micromolar [Ca(2+)] has no effect on PLB dynamics. A lipid anchor synthetically attached to the N terminus of PLB permits Ca-dependent SERCA inhibition but prevents the phosphorylation-induced disordering and reversal of inhibition. We conclude that the relief of SERCA inhibition by PLB phosphorylation is due to an order-to-disorder transition in the cytoplasmic domain of PLB, which allows this domain to extend above the membrane surface and induce a structural change in the cytoplasmic domain of SERCA. This mechanism is distinct from the one that relieves PLB-dependent SERCA inhibition upon the addition of micromolar [Ca(2+)].     

Phosphorylation of phospholamban in ischemia-reperfusion injury: Functional role of Thr17 residue

Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that when phosphorylated at Ser¹⁶ by PKA and/or at Thr¹⁷ by CaMKII increases the affinity of the SR Ca²⁺ pump for Ca²⁺. PLB is therefore, a critical regulator of SR function, myocardial relaxation and myocardial contractility. The present study was undertaken to examine the status of PLB phosphorylation after ischemia and reperfusion and to provide evidence about the possible role of the phosphorylation of Thr¹⁷ PLB residue on the recovery of contractility and relaxation after a period of ischemia. Experiments were performed in Langendorff perfused hearts from Wistar rats. Hearts were submitted to a protocol of global normothermic ischemia and reperfusion. The results showed that (1) the phosphorylation of Ser¹⁶ and Thr¹⁷ residues of PLB increased at the end of the ischemia and the onset of reperfusion, respectively. The increase in Thr¹⁷ phosphorylation was associated with a recovery of relaxation to preischemic values. This recovery occurred in spite of the fact that contractility was depressed. (2) The reperfusion-induced increase in Thr¹⁷ phosphorylation was dependent on Ca²⁺ entry to the cardiac cell. This Ca²⁺ influx would mainly occur by the coupled activation of the Na⁺/H⁺ exchanger and the Na⁺/Ca²⁺ exchanger working in the reverse mode, since phosphorylation of Thr¹⁷ was decreased by inhibition of these exchangers and not affected by blockade of the L-type Ca²⁺ channels. (3) Specific inhibition of CaMKII by KN93 significantly decreased Thr¹⁷ phosphorylation. This decrease was associated with an impairment of myocardial relaxation. The present study suggests that the phosphorylation of Thr¹⁷ of PLB upon reflow, may favor the full recovery of relaxation after ischemia. (Mol Cell Biochem 263: 131–136, 2004)     

Phosphorylation of phospholamban in ischemia-reperfusion injury: functional role of Thr17 residue

Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that when phosphorylated at Ser16 by PKA and/or at Thr17 by CaMKII increases the affinity of the SR Ca2+ pump for Ca2+. PLB is therefore, a critical regulator of SR function, myocardial relaxation and myocardial contractility. The present study was undertaken to examine the status of PLB phosphorylation after ischemia and reperfusion and to provide evidence about the possible role of the phosphorylation of Thr17 PLB residue on the recovery of contractility and relaxation after a period of ischemia. Experiments were performed in Langendorff perfused hearts from Wistar rats. Hearts were submitted to a protocol of global normothermic ischemia and reperfusion. The results showed that (1) the phosphorylation of Ser16 and Thr17 residues of PLB increased at the end of the ischemia and the onset of reperfusion, respectively. The increase in Thr17 phosphorylation was associated with a recovery of relaxation to preischemic values. This recovery occurred in spite of the fact that contractility was depressed. (2) The reperfusion-induced increase in Thr17 phosphorylation was dependent on Ca2+ entry to the cardiac cell. This Ca2+ influx would mainly occur by the coupled activation of the Na+ / H+ exchanger and the Na+ / Ca2+ exchanger working in the reverse mode, since phosphorylation of Thr17 was decreased by inhibition of these exchangers and not affected by blockade of the L-type Ca2+ channels. (3) Specific inhibition of CaMKII by KN93 significantly decreased Thr17 phosphorylation. This decrease was associated with an impairment of myocardial relaxation. The present study suggests that the phosphorylation of Thr17 of PLB upon reflow, may favor the full recovery of relaxation after ischemia.     

Heparin-derived oligosaccharides interact with the phospholamban cytoplasmic domain and stimulate SERCA function

The association between the cardiac transmembrane proteins phospholamban and sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a) regulates the active transport of Ca²⁺ into the sarcoplasmic reticulum (SR) lumen and controls the contraction and relaxation of the heart. Heart failure (HF) and cardiac hypertrophy have been linked to defects in Ca²⁺ uptake by the cardiac SR and stimulation of calcium transport by modulation of the PLB-SERCA interaction is a potential therapy. This work is part of an effort to identify compounds that destabilise the PLB-SERCA interaction in well-defined membrane environments. It is shown that heparin-derived oligosaccharides (HDOs) interact with the cytoplasmic domain of PLB and consequently stimulate SERCA activity. These results indicate that the cytoplasmic domain of PLB is functionally important and could be a valid target for compounds with drug-like properties.     

Structural changes in the cytoplasmic domain of phospholamban by phosphorylation at Ser16: a molecular dynamics study

Phospholamban is a 52-residue integral membrane protein that regulates the activity of the sarcoplasmic reticulum calcium pump in cardiac muscle. Its inhibitory action is relieved when phospholamban is phosphorylated at Ser16 by cAMP-dependent protein kinase. To computationally explore all possible conformations of the phosphorylated form, and thereby to understand the structural effects of phosphorylation, replica-exchange molecular dynamics (REMD) was applied to the cytoplasmic domain that includes Ser16. The simulations showed that (i) without phosphorylation, the region from Lys3 to Ser16 takes all alpha-helical conformations; (ii) when phosphorylated, the alpha-helix is partially unwound in the C-terminal part (from Ser10 to Ala15) resulting in less extended conformations; (iii) the phosphate at Ser16 forms salt bridges with Arg9, Arg13, and/or Arg14; and (iv) the salt bridges with Arg13 and Arg14 distort the alpha-helix and induce unwinding of the C-terminal part. We then applied conventional all-atom molecular dynamics simulations to the full-length phospholamban in the phospholipid bilayer. The results were consistent with those obtained with REMD simulations, suggesting that the transmembrane part of phospholamban and the lipid bilayer itself have only minor effects on the conformational changes in the cytoplasmic domain. The distortions caused by the salt bridges involving the phosphate at Ser16 readily explain the relief of the inhibitory effect of phospholamban by phosphorylation, as they will substantially reduce the population of all helical conformations, which are presumably required for the binding to the calcium pump. This will also be the mechanism for releasing the phosphorylated phospholamban from kinase.     

A conserved dimer and global conformational changes in the structure of apo-PknE Ser/Thr protein kinase from Mycobacterium tuberculosis

The "eukaryotic-like" receptor Ser/Thr protein kinases (STPKs) are candidates for the sensors that mediate environmental adaptations of Mycobacterium tuberculosis (Mtb). To define the mechanisms of regulation and substrate recognition, we determined the crystal structure of the ligand-free, activated kinase domain (KD) of the Mtb STPK, PknE. Remarkably, the PknE KD formed a dimer similar to that first observed in the structure of the ATPgammaS complex of the Mtb paralog, PknB. This structural similarity, which occurs despite little sequence conservation between the PknB and PknE dimer interfaces, supports the idea that dimerization regulates the Mtb receptor STPKs. Insertion of the DFG motif into the ATP-binding site and other conformational differences compared the ATPgammaS:PknB complex suggest that apo-PknE is not pre-organized to bind nucleotides. This structure may represent an inactive conformation stabilized by dimerization or, alternatively, an active conformation that reveals shifts that mediate nucleotide exchange and order substrate binding.     

Role of Mycobacterium tuberculosis Ser/Thr kinase PknF: implications in glucose transport and cell division

Protein kinases have a diverse array of functions in bacterial physiology, with a distinct role in the regulation of development, stress responses, and pathogenicity. pknF, one of the 11 kinases of Mycobacterium tuberculosis, encodes an autophosphorylating, transmembrane serine/threonine protein kinase, which is absent in the fast-growing, nonpathogenic Mycobacterium smegmatis. Herein, we investigate the physiological role of PknF using an antisense strategy with M. tuberculosis and expressing PknF and its kinase mutant (K41M) in M. smegmatis. Expression of PknF in M. smegmatis led to reduction in the growth rate and shortening and swelling of cells with constrictions. Interestingly, an antisense strain of M. tuberculosis expressing a low level of PknF displayed fast growth and a deformed cell morphology compared to the wild-type strain. Electron microscopy showed that most of the cells of the antisense strain were of a smaller size with an aberrant septum. Furthermore, nutrient transport analysis of these strains was conducted using 3H-labeled and 14C-labeled substrates. A significant increase in the uptake of D-glucose but not of glycerol, leucine, or oleic acid was observed in the antisense strain compared to the wild-type strain. The results suggest that PknF plays a direct/indirect role in the regulation of glucose transport, cell growth, and septum formation in M. tuberculosis.     

Intermolecular conformational coupling and free energy exchange enhance the catalytic efficiency of cardiac muscle SERCA2a following the relief of phospholamban inhibition

Activation of cardiac muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) by beta1-agonists involves cAMP- and PKA-dependent phosphorylation of phospholamban (PLB), which relieves the inhibitory effects of PLB on SERCA2a. To investigate the mechanism of SERCA2a activation, we compared the kinetic properties of SERCA2a expressed with (+) and without (-) PLB in High Five insect cell microsomes to those of SERCA1 and SERCA2a in native skeletal and cardiac muscle SR. Both native SERCA1 and expressed SERCA2a without PLB exhibited high-affinity (10-50 microM) activation of pre-steady-state catalytic site dephosphorylation by ATP, steady-state accumulation of the ADP-sensitive phosphoenzyme (E1P), and a rapid phase of EGTA-induced phosphoenzyme (E2P) hydrolysis. In contrast, SERCA2a in native cardiac SR vesicles and expressed SERCA2a with PLB lacked the high-affinity activation by ATP and the rapid phase of E2P hydrolysis, and exhibited low steady-state levels of E1P. The results indicate that the kinetic differences in Ca2+ transport between skeletal and cardiac SR are due to the presence of phospholamban in cardiac SR, and not due to isoform-dependent differences between SERCA1 and SERCA2a. Therefore, the results are discussed in terms of a model in which PLB interferes with SERCA2a oligomeric interactions, which are important for the mechanism of Ca2+ transport in skeletal muscle SERCA1 [Mahaney, J. E., Thomas, D. D., and Froehlich, J. P. (2004) Biochemistry 43, 4400-4416]. We propose that intermolecular coupling of SERCA2a molecules during catalytic cycling is obligatory for the changes in Ca2+ transport activity that accompany the relief of PLB inhibition of the cardiac SR Ca2+-ATPase.     

Dual Ser and Thr phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by MYPT-associated kinase

Phosphorylation of CPI-17 and PHI-1 by the MYPT1-associated kinase (M110 kinase) was investigated. M110 kinase is a recently identified serine/threonine kinase with a catalytic domain that is homologous to that of ZIP kinase (ZIPK. GST-rN-ZIPK, a constitutively active GST fusion fragment, phosphorylates CPI-17 (but not PHI-1) to a stoichiometry of 1.7 mol/mol. Phosphoamino acid analysis revealed phosphorylation of both Ser and Thr residues. Phosphorylation sites in CPI-17 were identified as Thr 38 and Ser 12 using Edman sequencing with (32)P release and a point mutant of Thr 38.     

Effects of Ser16 phosphorylation on the allosteric transitions of phospholamban/Ca(2+)-ATPase complex

Phosphorylation by protein kinase A and dephosphorylation by protein phosphatase 1 modulate the inhibitory activity of phospholamban (PLN), the endogenous regulator of the sarco(endo)plasmic reticulum calcium Ca(2+) ATPase (SERCA). This cyclic mechanism constitutes the driving force for calcium reuptake from the cytoplasm into the myocite lumen, regulating cardiac contractility. PLN undergoes a conformational transition between a relaxed (R) and tense (T) state, an equilibrium perturbed by the addition of SERCA. Here, we show that the single phosphoryl transfer at Ser16 induces a more pronounced conformational switch to the R state in phosphorylated PLN (pPLN). The binding affinity of PLN to SERCA is not affected (K(d) values for the transmembrane domains of pPLN and PLN are approximately 60 microM), supporting the hypothesis that phosphorylation at Ser16 does not dissociate PLN from SERCA. However, the binding surface and dynamics in domain Ib (residues 22-31) change substantially upon phosphorylation. Since PLN can be singly or doubly phosphorylated at Ser16 and Thr17, we propose that these sites remotely control the conformation of domain Ib. These findings constitute a paradigm for how post-translational modifications such as phosphorylation in the cytoplasmic portion of membrane proteins control intramembrane protein-protein interactions.     

Role of conformational sampling in computing mutation-induced changes in protein structure and stability

The prediction of changes in protein stability and structure resulting from single amino acid substitutions is both a fundamental test of macromolecular modeling methodology and an important current problem as high throughput sequencing reveals sequence polymorphisms at an increasing rate. In principle, given the structure of a wild-type protein and a point mutation whose effects are to be predicted, an accurate method should recapitulate both the structural changes and the change in the folding-free energy. Here, we explore the performance of protocols which sample an increasing diversity of conformations. We find that surprisingly similar performances in predicting changes in stability are achieved using protocols that involve very different amounts of conformational sampling, provided that the resolution of the force field is matched to the resolution of the sampling method. Methods involving backbone sampling can in some cases closely recapitulate the structural changes accompanying mutations but not surprisingly tend to do more harm than good in cases where structural changes are negligible. Analysis of the outliers in the stability change calculations suggests areas needing particular improvement; these include the balance between desolvation and the formation of favorable buried polar interactions, and unfolded state modeling.     

Increased inhibition of SERCA2 by phospholamban in the type I diabetic heart

The sarcoplasmic reticulum (SR) plays a critical role in mediating cardiac contractility and its function is abnormal in the diabetic heart. However, the mechanisms underlying SR dysfunction in the diabetic heart are not clear. Because protein phosphorylation regulates SR function, this study examined the phosphorylation state of phospholamban, a key SR protein that regulates SR calcium (Ca2+) uptake in the heart. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (STZ; 65 mg kg(-1) i.v.), and the animals were humanely killed after 6 weeks and cardiac SR function was examined. Depressed cardiac performance was associated with reduced SR Ca2+-uptake activity in diabetic animals. The reduction in SR Ca2+-uptake was consistent with a significant decrease in the level of SR Ca2+-pump ATPase (SERCA2a) protein. The level of phospholamban (PLB) protein was also decreased, however, the ratio of PLB to SERCA2a was increased in the diabetic heart. Depressed SR Ca2+-uptake was also due to a reduction in the phosphorylation of PLB by the Ca2+-calmodulin-dependent protein kinase (CaMK) and cAMP-dependent protein kinase (PKA). Although the activities of the SR-associated Ca2+-calmodulin-dependent protein kinase (CaMK), cAMP-dependent protein kinase (PKA) were increased in the diabetic heart, depressed phosphorylation of PLB could partly be attributed to an increase in the SR-associated protein phosphatase activities. These results suggest that there is increased inhibition of SERCA2a by PLB and this appears to be a major defect underlying SR dysfunction in the diabetic heart.     

Role of Ptc2 type 2C Ser/Thr phosphatase in yeast high-osmolarity glycerol pathway inactivation

Three type 2C Ser/Thr phosphatases (PTCs) are negative regulators of the yeast Saccharomyces cerevisiae high-osmolarity glycerol mitogen-activated protein kinase (MAPK) pathway. Ptc2 and Ptc3 are 75% identical to each other and differ from Ptc1 in having a noncatalytic domain. Previously, we showed that Ptc1 inactivates the pathway by dephosphorylating the Hog1 MAPK; Ptc1 maintains low basal Hog1 activity and dephosphorylates Hog1 during adaptation. Here, we examined the function of Ptc2 and Ptc3. First, deletion of PTC2 and/or PTC3 together with PTP2, encoding the protein tyrosine phosphatase that inactivates Hog1, produced a strong growth defect at 37°C that was dependent on HOG1, providing further evidence that PTC2 and PTC3 are negative regulators. Second, overexpression of PTC2 inhibited Hog1 activation but did not affect Hog1-Tyr phosphorylation, suggesting that Ptc2 inactivates the pathway by dephosphorylating the Hog1 activation loop phosphothreonine (pThr) residue. Indeed, in vitro studies confirmed that Ptc2 was specific for Hog1-pThr. Third, deletion of both PTC2 and PTC3 led to greater Hog1 activation upon osmotic stress than was observed in wild-type strains, although no obvious change in Hog1 inactivation during adaptation was seen. These results indicate that Ptc2 and Ptc3 differ from Ptc1 in that they limit maximal Hog1 activity. The function of the Ptc2 noncatalytic domain was also examined. Deletion of this domain decreased V_max by 1.6-fold and increased K_m by 2-fold. Thus Ptc2 requires an additional amino acid sequence beyond the catalytic domain defined for PTCs for full activity.     

Increased inhibition of SERCA2 by phospholamban in the type I diabetic heart

The sarcoplasmic reticulum (SR) plays a critical role in mediating cardiac contractility and its function is abnormal in the diabetic heart. However, the mechanisms underlying SR dysfunction in the diabetic heart are not clear. Because protein phosphorylation regulates SR function, this study examined the phosphorylation state of phospholamban, a key SR protein that regulates SR calcium (Ca²⁺) uptake in the heart. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (STZ; 65 mg kg^–1 i.v.), and the animals were humanely killed after 6 weeks and cardiac SR function was examined. Depressed cardiac performance was associated with reduced SR Ca²⁺-uptake activity in diabetic animals. The reduction in SR Ca²⁺-uptake was consistent with a significant decrease in the level of SR Ca²⁺-pump ATPase (SERCA2a) protein. The level of phospholamban (PLB) protein was also decreased, however, the ratio of PLB to SERCA2a was increased in the diabetic heart. Depressed SR Ca²⁺-uptake was also due to a reduction in the phosphorylation of PLB by the Ca²⁺-calmodulin-dependent protein kinase (CaMK) and cAMP-dependent protein kinase (PKA). Although the activities of the SR-associated Ca²⁺-calmodulin-dependent protein kinase (CaMK), cAMP-dependent protein kinase (PKA) were increased in the diabetic heart, depressed phosphorylation of PLB could partly be attributed to an increase in the SR-associated protein phosphatase activities. These results suggest that there is increased inhibition of SERCA2a by PLB and this appears to be a major defect underlying SR dysfunction in the diabetic heart. (Mol Cell Biochem 261: 245–249, 2004)     

Maximal inhibition of SERCA2 Ca(2+) affinity by phospholamban in transgenic hearts overexpressing a non-phosphorylatable form of phospholamban

Phospholamban is a phosphoprotein in the cardiac sarcoplasmic reticulum (SR) which regulates the apparent Ca(2+) affinity of the SR Ca(2+)-ATPase (SERCA2). To determine the levels of phospholamban which are associated with maximal inhibition of SERCA2, several lines of transgenic mice were generated which expressed increasing levels of a non-phosphorylatable form of phospholamban (S16A,T17A) specifically in the heart. This mutant form of phospholamban was chosen to prevent phosphorylation as a compensatory mechanism in vivo. Quantitative immunoblotting revealed increased phospholamban protein levels of 1.8-, 2.6-, 3.7-, and 4.7-fold in transgenic hearts compared with wild types. There were no changes in the expression levels of SERCA2, calsequestrin, calreticulin, and ryanodine receptor. Assessment of SR Ca(2+) uptake in hearts of transgenic mice indicated increases in the inhibition of the affinity of SERCA2 for Ca(2+) with increased phospholamban expression. Maximal inhibition was obtained at phospholamban expression levels of 2.6-fold or higher. Transgenic hearts with functional saturation in phospholamban:SERCA2 (>/=2.6:1) exhibited increases in beta-myosin heavy chain expression, associated with cardiac hypertrophy. These findings demonstrate that overexpression of a non-phosphorylatable form of phospholamban in transgenic mouse hearts resulted in saturation of the functional phospholamban:SERCA2 ratio at 2.6:1 and suggest that approximately 40% of the SR Ca(2+) pumps are functionally regulated by phospholamban in vivo.     

Protein phosphorylation on Ser, Thr and Tyr residues in cyanobacteria

Cyanobacteria belong to an extremely diverse group of gram-negative prokaryotes. They are all able to perform oxygen-evolving photosynthesis, but differ in morphology, ecological habitats, and physiology. This diversity is also reflected in the complexity of regulatory proteins involved in protein phosphorylation on Ser, Thr and Tyr residues. For those strains whose genomes are completely sequenced, for example, the number of genes identified so far that encode Ser/Thr and Tyr kinases range from none to 52. Genetic, molecular as well as functional genomic analyses demonstrate that Ser/Thr and Tyr kinases and phosphatases are involved in the regulation of a variety of activities according to changes in growth conditions or cell metabolism, such as cell motility, carbon and nitrogen metabolism, photosynthesis and stress response. The major challenge in the near future is to integrate these components into signaling pathways and identify their targets. Some of the Ser/Thr and Tyr kinases and phosphatases are expected to interact with classical two-component signaling pathways.     

Phosphorylation of Tau at Thr212, Thr231, and Ser262 Combined Causes Neurodegeneration

Abnormal hyperphosphorylation of the microtubule-associated protein Tau is a hallmark of Alzheimer disease and related diseases called tauopathies. As yet, the exact mechanism by which this pathology causes neurodegeneration is not understood. The present study provides direct evidence that Tau abnormal hyperphosphorylation causes its aggregation, breakdown of the microtubule network, and cell death and identifies phosphorylation sites involved in neurotoxicity. We generated pseudophosphorylated Tau proteins by mutating Ser/Thr to Glu and, as controls, to Ala. These mutations involved one, two, or three pathological phosphorylation sites by site-directed mutagenesis using as backbones the wild type or FTDP-17 mutant R406W Tau. Pseudophosphorylated and corresponding control Tau proteins were expressed transiently in PC12 and CHO cells. We found that a single phosphorylation site alone had little influence on the biological activity of Tau, except Thr²¹², which, upon mutation to Glu in the R406W background, induced Tau aggregation in cells, suggesting phosphorylation at this site along with a modification on the C-terminal of the protein facilitates self-assembly of Tau. The expression of R406W Tau pseudophosphorylated at Thr²¹², Thr²³¹, and Ser²⁶² triggered caspase-3 activation in as much as 85% of the transfected cells, whereas the corresponding value for wild type pseudophosphorylated Tau was 30%. Cells transfected with pseudophosphorylated Tau became TUNEL-positive.     

Role of active-site residues Thr81, Ser82, Thr85, Gln157, and Tyr158 in yeast cystathionine beta-synthase catalysis and reaction specificity

Cystathionine beta-synthase (CBS) effects the condensation of l-serine with l-homocysteine to form l-cystathionine. A series of active-site mutants, T81A, S82A, T85A, Q157A/E/H, and Y158F, was constructed to investigate effects on catalysis and reaction specificity in yeast CBS (yCBS). The effects of these mutations on the k(cat)/K(m)(L-Ser) for the beta-replacement reaction range from a reduction of only 3-fold for Y158F to below detectable levels for the Q157A and Q157E mutants. The order of importance of these residues to the beta-replacement reaction is Gln157 >or= Thr81 > Ser82 > Thr85 approximately Tyr158. All seven of the mutant enzymes catalyze a competing beta-elimination reaction, in which L-Ser is hydrolyzed to NH(3) and pyruvate. The ping-pong mechanism of CBS was thus expanded to include the latter reaction for these mutants. This activity is not detectable for wild-type yCBS, suggesting that the mutations result in a shift in the equilibrium between the open and the closed conformations of the active site of yCBS-substrate complexes. The Q157H and Y158F mutants additionally suffer suicide inhibition via a mechanism in which the released aminoacrylate intermediate covalently attacks the internal aldimine of the enzyme.