Calcium-dependent association of a protein complex with the lymphocyte plasma membrane: probable identity with calmodulin-calcineurin

A protein complex is shown to participate in a calcium-dependent association with plasma membranes purified either from pig mesenteric lymph node lymphocytes or from human lymphoblastoid cell lines. Plasma membranes prepared in the presence of calcium possess this complex; those prepared in the absence of calcium (5 mM EGTA) do not. The complex associates itself with the inner cytoplasmic surface of the plasma membrane. This complex is referred to as the "acidic protein band" because of its location during migration upon alkaline-urea gel electrophoresis. The complex dissociates from the plasma membrane during electrophoresis on 8-M urea gels, irrespective of calcium levels during electrophoresis; at intermediate urea concentrations (4-6 M), the complex is not dissociated in the presence of calcium. Upon purification of the acidic protein band, SDS acrylamide gel electrophoresis, immunoblotting, and radioimmunoassay techniques suggest that the acidic protein band is composed of at least four peptides (designated 68K, 59K, 20K, 20K): two of these (68K, 20K) are immunopositive for calcineurin and one (20K) is immunopositive for calmodulin. Immunoblots of urea gels also indicate that the calcineurin heavy chain (68K) can also appear at three different locations on the urea gel. Patches and caps induced in human peripheral blood lymphocytes by fluorescein-conjugated goat anti-human IgG are not coincident with the location of calcineurin, which remains distributed throughout the cell.     

The role of calpain in the selective increased phosphorylation of the anion-transport protein in red cell of hypertensive subjects

The phosphorylation of the anion-transport protein (band 3) is selectively increased in human red cell membrane, following exposure of intact cells to ionophore and micromolar calcium. The phosphorylation is catalyzed by a membrane associated protein kinase distinct from either protein kinase C or Ca2+/calmodulin dependent protein kinase. We show that the increase in phosphorylation of band 3 is abolished if red cells had been pre-loaded with an inhibitor of calpain or with an anticalpain monoclonal antibody. Our findings suggest that calpain activity may control, both at a functional and at a structural level, the activity of this important transmembrane protein through the modulation of its susceptibility as a substrate of membrane bound protein kinase(s). Based on previous observations indicating the presence in erythrocytes from hypertensive patients of an uncontrolled intracellular calpain-mediated proteolytic system accompanied by an increased phosphorylation of band 3 protein(s), we suggest that our results may shed light on the type of molecular alteration which is associated with the hypertensive state.     

Modulation of surface proteins by calcium in Chinese hamster lung cells

Investigations on the role of calcium in regulation of cell morphology of Chinese hamster lung cells (V79) revealed that cells grown with additional calcium (5 mM) in the growth medium (Ham's F12) adhere more tightly to the substratum than those grown in F12 alone. Additional calcium in the medium did not cause any changes in the structural membrane proteins or glycoproteins. Radioiodination of the surface membrane proteins of cells grown with or without additional calcium showed distinct differences in the labeling profile. The most striking change observed in cells grown with additional calcium was a very heavily labeled protein band at 70 K molecular weight. Two bands at approx. 100 K and 42 K were also heavily labeled. In contrast, the amount of radioactivity of a protein band at 52 K decreased in the cells grown in additional calcium. In general, cells grown with additional CaCl² were better iodinated than those grown in growth medium alone. The results demonstrate that calcium modulates surface proteins of V79 cells and this modulation may account for the changes observed in the cell morphology.     

Interaction between S100A8/A9 and annexin A6 is involved in the calcium-induced cell surface exposition of S100A8/A9

The calcium binding S100A8/A9 complex (MRP8/14; calgranulin) is considered as an important proinflammatory mediator in acute and chronic inflammation and has recently gained attention as a molecular marker up-regulated in various human cancers. Here, we report that S100A8/A9 is expressed in breast cancer cell lines and is up-regulated by interleukin-1beta and tumor necrosis factor-alpha in SKBR3 and MCF-7 cells. We identified the phospholipid-binding protein annexin A6 as a potential S100A8/A9 binding protein by affinity chromatography. This finding was verified by Southwestern overlay experiments and by coimmunoprecipitation with the S100A8/A9-specific monoclonal antibody 27E10. Immunocytochemical experiments demonstrated that S100A8/A9 and annexin A6 colocalize in SKBR3 breast cancer cells predominantly in membranous structures. Upon calcium influx both S100A8/A9 and annexin A6 are exposed on the cell surface of SKBR3 cells. Subcellular fractionation studies suggested that after A23187 stimulation membrane association of S100A8/A9 is not enhanced. However, both S100A8/A9 and annexin A6 are exposed on the cell surface of SKBR3 cells upon calcium influx. Experiments with artificial liposomes indicated that S100A8/A9 is able to associate with membranes independently of both annexin A6 and independently of calcium. Finally, cell surface expression of S100A8/A9 could not be observed in A23187-treated A431 and HaCaT cells. Both cell lines are known to be devoid of annexin A6. Repression of annexin A6 expression by small interfering RNA in SKBR3 cells abolishes the cell surface exposition of S100A8/A9 upon calcium influx, suggesting that annexin A6 contributes to the calcium-dependent cell surface exposition of the membrane associated-S100A8/A9 complex.     

Regulation of flagellar glycoprotein movements by protein phosphorylation

Cross-linking of surface exposed domains on certain Chlamydomonas flagellar membrane glycoproteins induces their movement within the plane of the flagellar membrane. A number of observations suggest that active movements of flagellar membrane glycoproteins are associated with the processes of whole cell gliding motility and the early events of fertilization in Chlamydomonas. Protein redistribution is totally inhibited if the free calcium concentration in the medium is 10(-7) M or below or in the presence of a number of calcium channel blockers (Bloodgood, R. A., N. L. Salomonsky, J. Cell Sci. 96, 27-33 (1990]. The present report demonstrates that glycoprotein redistribution in vivo is inhibited reversibly by three different protein kinase inhibitors: H-7, H-8 and staurosporine. Taken together, these observations suggest that the flagellum uses a signaling pathway that involves calcium influx induced by glycoprotein cross-linking, calcium activation of a protein kinase and specific protein phosphorylation to initiate flagellar surface dynamics.     

Casein binds to the cell membrane and induces intracellular calcium signals in the enteroendocrine cell: a brief communication

Dietary protein but not amino acids stimulates cholecystokinin (CCK) secretion in rat mucosal cells. However, the dietary protein sensory mechanisms and the intracellular signal pathway in the enteroendocrine cells have not yet been clarified. The relationship between dietary protein binding to cell membrane and intracellular calcium responses were examined in the CCK-producing enteroendocrine cell line STC-1. The binding of solubilized STC-1 cell membrane to proteins was analyzed using a surface plasmon resonance sensor. Intracellular calcium concentrations of STC-1 cell suspensions loaded with Fura-2 AM were measured using a spectrafluorophotometer system with continuous stirring. Intracellular calcium concentrations in STC-1 cells were increased by exposure to alpha-casein or casein sodium, but not to bovine serum albumin. Solubilized STC-1 membranes bound to alpha-casein and casein sodium but did not bind to bovine serum albumin. alpha-Casein demonstrated higher membrane binding and intracellular calcium stimulating activities than casein sodium. Thus, protein binding to the STC-1 cell membrane and intracellular calcium responses were correlated. Intracellular calcium responses to alpha-casein were suppressed by an L-type calcium channel blocker. These results suggest that casein, a dietary protein, binds to a putative receptor on the CCK-producing enteroendocrine cell membrane and elicits the subsequent intracellular calcium response via an L-type calcium channel.     

A Function of 40 kDa Outer Membrane Protein in Serratia marcescens

The function of one of the outer membrane proteins of Serratia marcescens was investigated. S. marcescens with an abundant 40 kDa outer membrane protein was induced to form spheroplast at a high rate in an isotonic medium in the presence of calcium, although the spheroplasts were generally fragile in the isotonic environment. The degree of spheroplast induction was correlated to the amount of the 40 kDa protein present in the membrane. In the 40 kDa proteinless mutant strains, the spheroplast induction rate was remarkably decreased. Autoradiography of the outer membrane revealed the presence of a calcium-binding protein as a radioactive band whose position coincided with the 40 kDa protein. These results suggest that the 40 kDa protein has an important role in maintaining the structural integrity of the cell wall against osmotic shock.     

Increased expression of a high molecular weight (130 KD) protein kinase C isoform in a differentiation-defective ras-transfected keratinocyte line

The role of ras on protein kinase C (PKC) signaling was examined in two keratinocyte cell lines. Increasing the level of extracellular calcium from 0.15 mM to 1.0 mM induces some features of differentiation in the spontaneously immortalized HaCaT line, but fails to do so in a c-H-ras-transfected subline (ras-HaCaT). Raising extracellular calcium also induced a transient increase in membrane-associated PKC activity 5 min after calcium addition, in HaCaT, but not in the ras-HaCaT cells. Partial purification of PKC from the membrane/particulate fraction revealed the major isoform expressed in HaCaT to be an 80 KD species recognized by the anti-PKCα antibody. In ras-HaCaT, the major expressed isoform is a 130 KD species recognized by the PKCb? antibody. The kinase activity of the partially purified high molecular weight PKC is phospholipid dependent but calcium independent. Further evaluation of PKC in the HaCaT and ras-HaCaT membrane/particulate cell fraction by immunoblotting using affinity-purified antibodies against PKCα, b?, δ, ε and ζ revealed a 130 KD band reacting with the PKCδ antibody. Increased expression of this high molecular weight protein was observed in ras-HaCaT. Immunoprecipitation of PKC in ras-HaCaT using the PKCδ antibody also revealed a 130 KD species. Analysis of the PKCδ immunoprecipitate demonstrated a phospholipid, but not calcium-dependent kinase which autophosphorylated. These results suggest that the 130 KD protein may be a novel (calcium-independent) PKC (nPKC) isoform and increased expression in the rastransfected HaCaT may be a consequence of oncogenic ras expression. This 130 KD species may also play a role in the ras-associated inhibition of differentiation in HaCaT. © 1995 Wiley-Liss, Inc.     

Erythrocyte aging and malaria.

The human malaria parasite, Plasmodium falciparum, ages the red blood cell during its intracellular development. During this process of erythrocyte senescence the parasitized cell becomes less dense and deformable, its biconcave disc shape becomes more spherical and is covered with microscopic protuberances (knobs); the amounts of membrane cholesterol and phospholipids are altered and phosphatidylserine (PS) is externalized. The malaria-infected cell is osmotically fragile, more permeable to a wide variety of molecules via new permeation pathways (NPP), and there is surface deposition of immunoglobulins and complement. There are declines in sialic acid, reduced glutathione, tocopherol and ATP. Hemichromes are deposited on the inner surface of the red cell membrane and there is clustering of the anion transporter, band 3 protein, as well as exposure of neoantigens which contribute to antigenic variation and adhesivity of the parasitized erythrocyte. These time-dependent changes result from oxidative assault and a combination of factors, including a decline in levels of anti-oxidants and ATP coupled with an enhanced flux of ions especially calcium. Despite these parasite-induced age effects P. falciparum is able to avoid destruction by splenic removal through microvessel sequestration in the deep tissues via PS, clustered band 3 protein and adhesive neoantigens.     

The structure and function of band 3 (AE1): Recent developments (Review)

This review discusses recent advances in our understanding of the structure, function and molecular genetics of the membrane domain of red cell anion exchanger, band 3 (AE1), and its role in red cell and kidney disease. A new model for the topology of band 3 has been proposed, which suggests the membrane domain has 12 membrane spans, rather than the 14 membrane spans of earlier models. The major difference between the models is in the topology of the region on the C-terminal side of membrane spans 1-7. Two dimensional crystals of the deglycosylated membrane domain of band 3 have yielded two and three dimensional projection maps of the membrane domain dimer at low resolution. The human band 3 gene has been completely sequenced and this has facilitated the study of natural band 3 mutations and their involvement in disease. About 20% of hereditary spherocytosis cases arise from heterozygosity for band 3 mutations, and result in the absence or decrease of the mutant protein in the red cell membrane. Several other natural band 3 mutations are known that appear to be clinically benign, but alter red cell phenotype or are associated with altered red cell blood group antigens. These include the mutant band 3 present in Southeast Asian ovalocytosis, a condition which provides protection against cerebral malaria in children. Familial distal renal tubular acidosis, a condition associated with kidney stones, has been shown to result from a novel group of band 3 mutations. The total absence of band 3 has been described in animals-occurring naturally in cattle and after targeted disruption in mice. Some of these severely anaemic animals survive, so band 3 is not strictly essential for life. Although the band 3-negative red cells were very unstable, they contained a normally-assembled red cell skeleton, suggesting that the bilayer of the normal red cell membrane is stabilized by band 3 interactions with membrane lipids, rather than by interactions with the spectrin skeleton.     

Phosphorylation sites in human erythrocyte band 3 protein

The human red cell anion-exchanger, band 3 protein, is one of the main phosphorylated proteins of the erythrocyte membrane. Previous studies from this laboratory have shown that ATP-depletion of the red blood cell decreased the anion-exchange rate, suggesting that band 3 protein phosphorylation could be involved in the regulation of anion transport function (Bursaux et al. (1984) Biochim. Biophys. Acta 777, 253-260). Phosphorylation occurs mainly on the cytoplasmic domain of the protein and the major site of phosphorylation was assigned to tyrosine-8 (Dekowski et al. (1983) J. Biol. Chem. 258, 2750-2753). This site being very far from the integral, anion-exchanger domain, the aim of the present study was to determine whether phosphorylation sites exist in the integral domain. The phosphorylation reaction was carried out on isolated membranes in the presence of [gamma-32P]ATP and phosphorylated band 3 protein was then isolated. Both the cytoplasmic and the membrane spanning domains were purified. The predominant phosphorylation sites were found on the cytoplasmic domain. RP-HPLC analyses of the tryptic peptides of whole band 3 protein, and of the isolated cytoplasmic and membrane-spanning domains allowed for the precise localization of the phosphorylated residues. 80% of the label was found in the N-terminal tryptic peptide (T-1), (residues 1-56). In this region, all the residues susceptible to phosphorylation were labeled but in varying proportion. Under our conditions, the most active membrane kinase was a tyrosine kinase, activated preferentially by Mn2+ but also by Mg2+. Tyrosine-8 was the main phosphate acceptor residue (50-70%) of the protein, tyrosine-21 and tyrosine-46 residues were also phosphorylated but to a much lesser extent. The main targets of membrane casein kinase, preferentially activated by Mg2+, were serine-29, serine-50, and threonine(s)-39, -42, -44, -48, -49, -54 residue(s) located in the T-1 peptide. A tyrosine phosphatase activity was copurified with whole band 3 protein which dephosphorylates specifically P-Tyr-8, indicating a highly exchangeable phosphate. The membrane-spanning fragment was only faintly labeled.     

Filamin translocation is an early endothelial cell inflammatory response to bradykinin: Regulation by calcium,protein kinases,and protein phosphatases

Endothelial cell (EC) cytoskeletal proteins are one of the earliest primary targets of second messenger cascades generated in response to inflammatory agonists. Actin binding proteins, by modulating actin gelation-solation state and membrane-cytoskeleton interactions, in part regulate cell motility and cell-cell apposition. This in turn can also modulate interendothelial junctional diameter and permeability. Nonmuscle filamin (ABP-280), a dimeric actincrosslinking protein, promotes orthogonal branching of F-actin and links microfilaments to membrane glycoproteins. In the present study, immunoblot analysis demonstrates that filamin protein levels are low in sparse EC cultures, increase once cell-cell contact is initiated and then decrease slightly at post-confluency. Both bradykinin and ionomycin cause filamin redistribution from the peripheral cell border to the cytosol of confluent EC. Forskolin, an activator of adenylate cyclase, blocks filamin translocation. Bradykinin activation of EC is not accompanied by significant proteolytic cleavage of filamin. Instead, intact filamin is recycled back to the membrane within 5–10 min of bradykinin stimulation. Inhibitors of calcium/calmodulin dependent protein kinase (KT-5926 and KN-62) attenuate bradykinin-induced filamin translocation. H-89, an inhibitor of cAMP-dependent protein kinase, causes translocation of filamin in unstimulated cells. Calyculin A, an inhibitor of protein phosphatases, also causes translocation of filamin in the absence of an inflammatory agent. ML-7, an inhibitor of myosin light chain kinase and phorbol myristate acetate, an activator of protein kinase C, do not cause filamin movement into the cytosol, indicating that these pathways do not modulate the translocation. Pharmacological data suggest that filamin translocation is initiated by the calcium/calmodulin-dependent protein kinase whereas the cAMP-dependent protein kinase pathway prevents translocation. Inflammatory agents therefore may increase vascular junctional permeability by increasing cytoplasmic calcium, which disassembles the microfilament dense peripheral band by releasing filamin from F-actin. © 1996 Wiley-Liss, Inc.     

Bay K8644 like activity of an antibody against a 60 kDa tubular membrane protein

Partial purification of the dihydropyridine receptor from rat skeletal muscle demonstrated mainly a 60 kDa band in SDS-polyacrylamide gel. An antibody raised against that protein behaved as a calcium channel agonist viz. Bay K8644. The affinity purified antibody, when added to cultured heart cells, increased the beat rate 40-80% depending on the titer of the antiserum. The antibody also woke up the beats of the cells previously blocked with the channel antagonist, nifedipine. Immunoblot analysis indicated that the receptor of this antibody in heart cell membrane is also a 60 kDa protein.     

Rapid Changes in Plasma Membrane Protein Phosphorylation during Initiation of Cell Wall Digestion

Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with [γ-³²P]ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M_r 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M_r 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M_r 22,000. These effects appeared not to be due to nonspecific protease activity and neither in vivo nor in vitro exposure to driselase caused a significant loss of Coomassie blue-staining bands on the gels of the isolated plasma membranes. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic ³²P also showed a response to the Driselase treatment. An enzymically active driselase preparation was required for the observed responses.     

Limited proteolysis of the erythrocyte membrane skeleton by calcium-dependent proteinases

The action of purified calcium-dependent proteinases on human erythrocyte membrane skeleton proteins has been examined. Preferential cleavage of proteins 4.1 a and b and band 3 and limited cleavage of alpha- and beta-spectrin occur when either calcium-dependent proteinase I or calcium-dependent proteinase II has access to the cytoplasmic side of the ghost membrane skeleton in the presence of calcium. Thus, when these proteinases are incubated with sealed ghosts they do not cleave these proteins. Leupeptin, mersalyl, the specific cellular protein inhibitor of these enzymes, and calcium chelators can inhibit proteolysis of the red cell ghost proteins by Ca2+-dependent proteinases. Each proteinase has also been loaded into erythrocyte ghosts in the absence of calcium at low ionic strength and subsequently trapped inside by resealing the ghosts. The proteinases were activated by incubating these ghosts in the presence of the calcium ionophore A23187 and calcium. Examination of the ghost proteins by electrophoresis demonstrated calcium-dependent proteolysis of Bands 4.1 and 3 and limited cleavage of alpha- and beta-spectrin similar to that observed on proteolysis of the open, leaky ghosts. In the presence of calcium each calcium-dependent proteinase appears to associate with the erythrocyte ghost membrane.     

Calcium homeostasis and cell death in Sol8 dystrophin-deficient cell line in culture

Abnormalities of calcium homeostasis are involved in the process of cell injuries such as Duchenne muscular dystrophy characterized by the absence of the protein dystrophin. But how the absence of dystrophin leads to cytosolic calcium overload is as yet poorly understood. This question has been addressed with skeletal muscle cells from human DMD muscles or mdx mice. Although easier to obtain than human muscles, mdx muscle cells have provided controversial data concerning the resting intracellular calcium level ([Ca2+](i)). This work describes the culture of Sol8 cell line that expresses neither dystrophin nor adhalin, a dystrophin-associated protein. The [Ca2+](i)and intracellular calcium transients induced by different stimuli (acetylcholine, caffeine and high potassium) are normal during the first days of culture. At later stages, calcium homeostasis exhibits drastic alterations with a breaking down of the calcium responses and a large [Ca2+](i)elevation. Concomitantly, Sol8 cells exhibit morphological signs of cell death like cytoplasmic shrinkage and incorporation of propidium iodide. Cell death could be significantly reduced by blocking the activity of calpains, a type of calcium-regulated proteases. These results suggest that Sol8 cell line provides an alternative model of dystrophin-deficient skeletal muscle cells for which a clear disturbance of the calcium homeostasis is observed in culture in association with calpain-dependent cell death. It is shown that transfection with a plasmid cDNA permits the forced expression of dystrophin in Sol8 myotubes as well as a correct sorting of the protein. This approach could be used to explore possible interactions between dystrophin deficiency, calcium homeostasis alteration, and dystrophic cell death.     

Band 3 tyr-phosphorylation in human erythrocytes from non-pregnant and pregnant women

Pregnancy is associated with changes in circulating red blood cells, mainly involving band 3 protein and membrane lipid peroxidation. Membrane band 3 is a multifunctional protein containing four Tyr-phosphorylatable residues which modulate the physiological status of erythrocytes by regulating glycolysis, cell shape and membrane transport. Erythrocytes from nine pregnant and 12 age-matched non-pregnant healthy women were subjected to oxidative and hyperosmotic stress conditions and the extent of band 3 Tyr-phosphorylation and membrane Syk recruitment as a membrane marker were evaluated. Results indicated that, in pregnancy, red blood cells show a decrease in band 3 Tyr-phosphorylation and a clear-cut rearrangement of band 3 protein within the membrane. In fact, band 3 shows a decrease in high molecular weight aggregates (HMWA), with different subdivision between Triton-soluble and -insoluble compartments, and an increase in proteolytic fragments. In conclusion, it is demonstrated that pregnancy is associated with membrane adjustments which reduce the sensitivity of erythrocytes to both oxidative and osmotic stress. Band 3 Tyr-phosphorylation is proposed as a new parameter in the evaluation of erythrocyte membrane arrangement.     

Band 3 is a target protein of spectrin's E2/E3 activity: implication for sickle cell disease and normal red blood cell aging.

We have demonstrated that a 125 kDa red blood cell (RBC) membrane protein, being a target of spectrin's E2/E3 activity, is ubiquitinated band 3. This demonstration was based on copurification of this biotinylated-ubiquitinated protein with band 3, immunoprecipitation with band 3 antibody and analysis of proteins associated with strepavidin sepharose by micro liquid chromatography coupled to tandem mass spectrometry (microLC/MS/MS). Further, we demonstrated the presence of ubiquitinated band 3 in vivo by Western blotting of purified band 3 with a monoclonal antibody (FK2) against ubiquitin. The implications of these results for sickle cell disease and RBC aging are discussed.     

Band 3 tyr-phosphorylation in human erythrocytes from non-pregnant and pregnant women

Pregnancy is associated with changes in circulating red blood cells, mainly involving band 3 protein and membrane lipid peroxidation. Membrane band 3 is a multifunctional protein containing four Tyr-phosphorylatable residues which modulate the physiological status of erythrocytes by regulating glycolysis, cell shape and membrane transport. Erythrocytes from nine pregnant and 12 age-matched non-pregnant healthy women were subjected to oxidative and hyperosmotic stress conditions and the extent of band 3 Tyr-phosphorylation and membrane Syk recruitment as a membrane marker were evaluated. Results indicated that, in pregnancy, red blood cells show a decrease in band 3 Tyr-phosphorylation and a clear-cut rearrangement of band 3 protein within the membrane. In fact, band 3 shows a decrease in high molecular weight aggregates (HMWA), with different subdivision between Triton-soluble and -insoluble compartments, and an increase in proteolytic fragments. In conclusion, it is demonstrated that pregnancy is associated with membrane adjustments which reduce the sensitivity of erythrocytes to both oxidative and osmotic stress. Band 3 Tyr-phosphorylation is proposed as a new parameter in the evaluation of erythrocyte membrane arrangement.     

Calcium-Actin Waves and Oscillations of Cellular Membranes

We propose a mechanism for the formation of membrane oscillations and traveling waves, which arise due to the coupling between the actin cytoskeleton and the calcium flux through the membrane. In our model, the fluid cell membrane has a mobile but constant population of proteins with a convex spontaneous curvature, which act as nucleators of actin polymerization and adhesion. Such a continuum model couples the forces of cell-substrate adhesion, actin polymerization, membrane curvature, and the flux of calcium through the membrane. Linear stability analysis shows that sufficiently strong coupling among the calcium, membrane, and protein dynamics may induce robust traveling waves on the membrane. This result was checked for a reduced feedback scheme and is compared to the results without the effects of calcium, where permanent phase separation without waves or oscillations is obtained. The model results are compared to the published observations of calcium waves in cell membranes, and a number of testable predictions are proposed.