Regulation of Ca2؉ /calmodulin-dependent protein kinase II is likely based on an auto-inhibitory mechanism in which a segment of the kinase occupies the catalytic site in the absence of calmodulin. We analyze potential auto-inhibitory associations by employing charge reversal and hydrophobic-to-charged residue mutagenesis. We identify interacting amino acid pairs by using double mutants to test which modification in the catalytic domain complements a given change in the auto-inhibitory domain. Our studies identify the core pseudosubstrate sequence (residues 297-300) but reveal that distinct sequences centered about the autophosphorylation site at Thr-286 are involved in the critical auto-inhibitory interactions. Individual changes in any of the residues Arg-274, His-282, Arg-283, Lys-291, Arg-297, Phe-293, and Asn-294 in the auto-inhibitory domain or their interacting partners in the catalytic domain produces an enhanced affinity for calmodulin or generates a constitutively active enzyme. A structural model of Ca 2؉ /calmodulin-dependent protein kinase II that incorporates these interactions shows that Thr-286 is oriented inwardly into a hydrophobic channel. The model explains why calmodulin must bind to the auto-inhibitory domain in order for Thr-286 in that domain to be phosphorylated and why introduction of phospho-Thr-286 produces the important Ca 2؉ -independent state of the enzyme.Multifunctional Ca 2ϩ /calmodulin-dependent protein kinase (CaM kinase) 1 II has received considerable attention because of its autoregulatory properties (reviewed in Refs. 1 and 2). Regulation of the kinase is likely based on an auto-inhibitory or pseudosubstrate mechanism in which a segment of the kinase occupies the catalytic site in the basal state. Ca 2ϩ /calmodulin activates the kinase by wrapping around its target sequence on the kinase, a site that overlaps the auto-inhibitory domain (3). The active enzyme not only phosphorylates exogenous substrates but also exhibits a prominent autophosphorylation of Thr-286 within the auto-inhibitory domain. Autophosphorylation is an intersubunit reaction occurring within each holoenzyme and requires that calmodulin activate one subunit serving as kinase, while a second calmodulin is bound to the subunit serving as substrate (4 -6). Autophosphorylation traps bound calmodulin by greatly reducing its dissociation rate and prolonging the active state (7). Even after calmodulin dissociates, the autophosphorylated kinase remains partially active or autonomous of Ca 2ϩ /calmodulin (8 -11). Phospho-Thr-286 may therefore be positioned to interfere with re-establishment of auto-inhibitory contacts. This autophosphorylation is critical for Ca 2ϩ spike frequency-dependent activation of the enzyme (12) and enhances targeting of the kinase to synaptic sites (13-15) that may underlie the role of the kinase in regulation of synaptic strength. Mice defective in this autophosphorylation (␣-CaM kinase II Thr-286 3 Ala mutants) do not exhibit long term potentiation and are defective in spatial learning (...

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Alteration of red blood cell (RBC) pH produces stomatocytosis (at low pH) and echinocytosis (at high pH). Cell shrinkage potentiates high pH echinocytosis, but shrinkage alone does not cause echinocytosis. Mechanisms for these shape changes have not been described. In this study, measured dependence of RBC shape on cell pH was nonlinear, with a broad pH range in which normal discoid shape was maintained. Transbilayer distribution of phosphatidylcholine and phosphatidylserine, measured by back-extraction of radiolabeled lipid, was the same in control and altered pH cells. Possible roles of pH- titratable inner monolayer phospholipids were examined by assessing pH- dependent shape in cells in which their levels had been perturbed. In metabolically depleted cells and calcium-treated cells, which have altered levels of phosphatidic acid, phosphatidylinositol-4-phosphate, and/or phosphatidylinositol-4,5-bisphosphate, low cell pH was stomatocytogenic and high cell pH was echinocytogenic, as in control cells. Thus, neither change in membrane lipid asymmetry nor normal levels of the pH-titratable inner monolayer lipids is necessary for cell pH-mediated shape change.

Echinocytosis induced by elevation of intracellular Ca2+ in human erythrocytes can be reversed by removal of the cation. Using back-extraction of radiolabeled dilauroyl phospholipid analogs which had been incorporated into the cell membrane, we examined the relationship between this reversible shape transformation and phospholipid distribution. Upon Ca2+ crenation of cells, surface exposure of phosphatidylserine and phosphatidylethanolamine was observed simultaneously with inward diffusion of phosphatidylcholine. Removal of Ca2+ allowed resequestration of exposed phosphatidylserine to the membrane inner monolayer, but randomized phosphatidylethanolamine and phosphatidylcholine were not redistributed to their original states. Both shape reversion and retranslocation of phosphatidylserine were reversibly inhibited vanadate. On the other hand, the cell shape recovery was found to be independent of membrane skeleton and phosphoinositide metabolism and was supported by ATP resynthesis only under conditions where the aminophospholipid translocator is active. Other Ca(2+)-mediated biochemical changes, such as generation of diacylglycerol and fatty acids, were found to have no effect on Ca2+ crenation or its reversal, or upon transbilayer distribution of any phospholipid. These findings suggest that Ca2+ induces phospholipid redistribution, possibly by direct interaction with the lipid bilayer and, further, that metabolic recovery from Ca2+ crenation reflects selective retransport of phosphatidylserine to the membrane inner monolayer.

The molecular basis of cell shape regulation in acidic pH was investigated in human erythrocytes. Intact erythrocytes maintain normal shape in the cell pH range 6.3-7.9, but invaginate at lower pH values. However, consistent with predicted pH-dependent changes in the erythrocyte membrane skeleton, isolated erythrocyte membranes evaginate in acidic pH. Moreover, intact cells evaginate at pH greater than 7.9, but isolated membranes invaginate in this condition. Labeling with the hydrophobic, photoactivatable probe 5-[125I]iodonaphthyl-1-azide demonstrated pH-dependent hydrophobic insertion of an amphitropic protein into membranes of intact cells but not into isolated membranes. Based on molecular weight and on reconstitution experiments using stripped inside-out vesicles, the most likely candidate for the variably labeled protein is glyceraldehyde-3-phosphate dehydrogenase. Resealing of isolated membranes reconstituted both the shape changes and the hydrophobic labeling profile seen in intact cells. This observation appears to resolve the paradox of the contradictory pH dependence of shape changes of intact cells and isolated membranes. In intact erythrocytes, the demonstrated protein-membrane interaction would oppose pH-dependent shape effects of the spectrin membrane skeleton, stabilizing cell shape in moderately abnormal pH. Stabilization of erythrocyte shape in moderately acidic pH may prevent inappropriate red cell destruction in the spleen.