Modulation of Erythrocyte Membrane Mechanical Function by Protein 4.1 Phosphorylation

Erythrocyte membrane mechanical function is regulated by the spectrin-based membrane skeleton composed of α- and β-spectrin, actin, protein 4.1R (4.1R), and adducin. Post-translational modifications of these proteins have been suggested to modulate membrane mechanical function. Indeed, β-spectrin phosphorylation by casein kinase I has been shown to decrease membrane mechanical stability. However, the effects of the phosphorylation of skeletal proteins by protein kinase C (PKC), a serine/threonine kinase, have not been elucidated. In the present study, we explored the functional consequences of the phosphorylation of 4.1R and adducin by PKC. We identified Ser-312 in 4.1R as the PKC phosphorylation site. Using antibodies raised against phosphopeptides of 4.1R and adducin, we documented significant differences in the time course of phosphorylation of adducin and 4.1R by PKC. Although adducin was phosphorylated rapidly by the activation of membrane-bound atypical PKC by phorbol 12-myristate 13-acetate stimulation, there was a significant delay in the phosphorylation of 4.1R because of delayed recruitment of conventional PKC from cytosol to the membrane. This differential time course in the phosphorylation of 4.1R and adducin in conjunction with membrane mechanical stability measurements enabled us to document that, although phosphorylation of adducin by PKC has little effect on membrane mechanical stability, additional phosphorylation of 4.1R results in a marked decrease in membrane mechanical stability. We further showed that the phosphorylation of 4.1R by PKC results in its decreased ability to form a ternary complex with spectrin and actin as well as dissociation of glycophorin C from the membrane skeleton. These findings have enabled us to define a regulatory role for 4.1R phosphorylation in dynamic regulation of red cell membrane properties. Erythrocyte membrane mechanical function is regulated by the spectrin-based membrane skeleton composed of α- and β-spectrin, actin, protein 4.1R (4.1R), and adducin. Post-translational modifications of these proteins have been suggested to modulate membrane mechanical function. Indeed, β-spectrin phosphorylation by casein kinase I has been shown to decrease membrane mechanical stability. However, the effects of the phosphorylation of skeletal proteins by protein kinase C (PKC), a serine/threonine kinase, have not been elucidated. In the present study, we explored the functional consequences of the phosphorylation of 4.1R and adducin by PKC. We identified Ser-312 in 4.1R as the PKC phosphorylation site. Using antibodies raised against phosphopeptides of 4.1R and adducin, we documented significant differences in the time course of phosphorylation of adducin and 4.1R by PKC. Although adducin was phosphorylated rapidly by the activation of membrane-bound atypical PKC by phorbol 12-myristate 13-acetate stimulation, there was a significant delay in the phosphorylation of 4.1R because of delayed recruitment of conventional PKC from cytosol to the membrane. This differential time course in the phosphorylation of 4.1R and adducin in conjunction with membrane mechanical stability measurements enabled us to document that, although phosphorylation of adducin by PKC has little effect on membrane mechanical stability, additional phosphorylation of 4.1R results in a marked decrease in membrane mechanical stability. We further showed that the phosphorylation of 4.1R by PKC results in its decreased ability to form a ternary complex with spectrin and actin as well as dissociation of glycophorin C from the membrane skeleton. These findings have enabled us to define a regulatory role for 4.1R phosphorylation in dynamic regulation of red cell membrane properties. The maintenance of normal membrane deformability and mechanical stability is critical for human red blood cells to undergo extensive deformations in the microvasculature, which is necessary to perform their function of oxygen delivery during their 120-day life span. The well characterized spectrin-based membrane skeleton, composed of α- and β-spectrin, actin, protein 4.1R (4.1R), adducin, dematin, tropomyosin, and tropomodulin, plays a critical role in regulating membrane mechanical function (1Mohandas N. Chasis J.A. Semin. Hematol. 1993; 30: 171-192PubMed Google Scholar). Qualitative and quantitative defects in α- and β-spectrin and 4.1R that lead either to an impaired spectrin-spectrin self-association or to a weakened spectrin-actin-4.1R ternary complex have been shown to result in decreased membrane mechanical stability (2Tse W.T. Lecomte M.C. Costa F.F. Garbarz M. Feo C. Boivin P. Dhermy D. Forget B.G. J. Clin. Investig. 1990; 86: 909-916Crossref PubMed Scopus (88) Google Scholar, 3Gallagher P.G. Tse W.T. Coetzer T. Lecomte M.C. Garbarz M. Zarkowsky H.S. Baruchel A. Ballas S.K. Dhermy D. Palek J. Forget B.G. J. Clin. Investig. 1992; 89: 892-898Crossref PubMed Scopus (28) Google Scholar, 4Tchernia G. Mohandas N. Shohet S.B. J. Clin. Investig. 1981; 68: 454-460Crossref PubMed Scopus (133) Google Scholar, 5Takakuwa Y. Tchernia G. Rossi M. Benabadji M. Mohandas N. J. Clin. Investig. 1986; 78: 80-85Crossref PubMed Scopus (87) Google Scholar). A number of studies over the years have documented that skeletal proteins β-spectrin, 4.1R, adducin, and dematin can be phosphorylated by a number of different kinases and are dephosphorylated by various phosphatases (6Boivin P. Biochem. J. 1988; 256: 689-695Crossref PubMed Scopus (58) Google Scholar, 7Cohen C.M. Gascard P. Semin. Hematol. 1992; 29: 244-292PubMed Google Scholar). In vitro studies using purified proteins have shown that phosphorylation can alter skeletal protein function (6Boivin P. Biochem. J. 1988; 256: 689-695Crossref PubMed Scopus (58) Google Scholar, 7Cohen C.M. Gascard P. Semin. Hematol. 1992; 29: 244-292PubMed Google Scholar). However, our understanding of how the phosphorylation of various red cell membrane skeletal proteins regulates the mechanical function of intact red cell membranes has not been well defined. In a previous study, we documented that phosphorylation of β-spectrin by casein kinase I decreases the mechanical stability of intact membranes (8Manno S. Takakuwa Y. Nagao K. Mohandas N. J. Biol. Chem. 1995; 270: 5659-5665Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). 4.1R and adducin form a ternary protein complex with spectrin and actin, and both of them promote the association of spectrin with actin filaments (9Ungewickell E. Bennett V. Calvert R. Ohanian V. Gratzer W.B. Nature. 1979; 280: 811-814Crossref PubMed Scopus (150) Google Scholar, 10Gardner K. Bennett V. Nature. 1987; 328: 359-362Crossref PubMed Scopus (200) Google Scholar, 11Bennett V. Gardner K. Steiner J.P. J. Biol. Chem. 1988; 263: 60-5869Google Scholar, 12Li X. Matsuoka Y. Bennett V. J. Biol. Chem. 1998; 273: 19329-19338Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 13Matsuoka Y. Li X. Bennett V. CMLS Cell. Mol. Life Sci. 2000; 57: 884-895Crossref PubMed Scopus (252) Google Scholar). 4.1R also binds to the integral membrane proteins glycophorin C (GPC) 1The abbreviations used are: GPC, glycophorin C; SAB, spectrinactin binding; PK, protein kinase; IOV, inside-out vesicle; PMA, phorbol 12-myristate 13-acetate; aPKC, atypical PKC; cPKC, conventional PKC; P-4.1R, phosphorylated protein 4.1R; PVDF, polyvinylidene difluoride; TBS, Tris-buffered saline; DI, deformability index; FERM, 4.1-ezrin-radixin-moesin. and band 3. Although an important role for 4.1R in regulating membrane mechanical stability has been documented (5Takakuwa Y. Tchernia G. Rossi M. Benabadji M. Mohandas N. J. Clin. Investig. 1986; 78: 80-85Crossref PubMed Scopus (87) Google Scholar), the role of adducin in regulating membrane mechanical function has yet to be delineated. Limited chymotryptic digestion of 4.1R generates four distinct polypeptides, the 30-, 16-, 10-, and 22/24-kDa fragments (14Conboy J.G. Semin. Hematol. 1993; 30: 58-73PubMed Google Scholar). The 30-kDa N-terminal domain, referred to as the membrane binding or FERM domain (15Chishti A.H. Kim A.C. Marfatia S.M. Lutchman M. Hanspal M. Jindal H. Liu S.C. Low P.S. Rouleau G.A. Mohandas N. Chasis J.A. Conboy J.G. Gascard P. Takakuwa Y. Huang S.C. Benz Jr., E.J. Bretscher A. Fehon R.G. Gusella J.F. Ramesh V. Solomon F. Marchesi V.T. Tsukita S. Tsukita S. Arpin M. Louvard D. Tonks N.K. Anderson J.M. Fanning A.S. Bryant P.J. Woods D.F. Hoover K.B. Trends Biochem. 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Conboy J.G. Mohandas N. Snyder S.H. J. Cell Biol. 1998; 141: 143-153Crossref PubMed Scopus (107) Google Scholar), nuclear mitotic apparatus protein (NuMA) (28Mattagajasingh S.N. Huang S.C. Hartenstein J.S. Snyder M. Marchesi V.T. Benz E.J. J. Cell Biol. 1999; 145: 29-43Crossref PubMed Scopus (112) Google Scholar), and zonula occluden-2 (ZO-2) (29Mattagajasingh S.N. Huang S.C. Hartenstein J.S. Benz Jr., E.J. J. Biol. Chem. 2000; 275: 30573-30585Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). 4.1R can be phosphorylated both in vitro and in vivo by protein kinase A (PKA) and PKC (21Danilov Y.N. Fennell R. Ling E. Cohen C.M. J. Biol. Chem. 1990; 265: 2556-2562Abstract Full Text PDF PubMed Google Scholar, 30Ling E. Danilov Y.N. Cohen C.M. J. Biol. Chem. 1988; 263: 2209-2216Abstract Full Text PDF PubMed Google Scholar, 31Pinder J.C. Gardner B. Gratzer W.B. Biochem. Biophys. Res. Commun. 1995; 210: 478-482Crossref PubMed Scopus (16) Google Scholar, 32Horne W.C. Leto T.L. Marchesi V.T. J. Biol. Chem. 1985; 260: 9073-9076Abstract Full Text PDF PubMed Google Scholar, 33Horne W.C. Prinz W.C. Tang E.K. Biochim. Biophys. Acta. 1990; 1055: 87-92Crossref PubMed Scopus (22) Google Scholar). Previous studies (30Ling E. Danilov Y.N. Cohen C.M. J. Biol. Chem. 1988; 263: 2209-2216Abstract Full Text PDF PubMed Google Scholar) have documented that in solution, PKA the of and of purified 4.1R that the phosphorylation of these has effect on the ability of 4.1R to interact with its red cell membrane binding However, phosphorylation of 4.1R by a yet to be decreases the ability of 4.1R to promote spectrin-actin and its ability to to the domain of band 3 in inside-out has effect on 4.1R binding to GPC (21Danilov Y.N. Fennell R. Ling E. Cohen C.M. J. Biol. Chem. 1990; 265: 2556-2562Abstract Full Text PDF PubMed Google Scholar). In a J.C. Gardner B. Gratzer W.B. Biochem. Biophys. Res. Commun. 1995; 210: 478-482Crossref PubMed Scopus (16) Google Scholar), phorbol 12-myristate 13-acetate of red blood cells has been shown to result in a decreased association of GPC with the membrane skeleton, that PKC phosphorylation GPC from its skeletal The effect of the phosphorylation of 4.1R on the mechanical function of an intact membrane has yet to be defined. is present as a of in a in the red cell L. C. B. L. K. S. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). are in and of them an N-terminal domain, a domain, and a C-terminal domain R. Bennett V. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar). The domain of adducin with a protein kinase C domain and the PKC phosphorylation site. In of and of have been shown Y. Bennett V. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Li X. Bennett V. J. Cell Biol. 1998; PubMed Scopus Google Scholar) to be phosphorylated by PKC in However, the effect of phosphorylation of adducin on membrane mechanical function has yet to be defined. In the present study, we explored the functional consequences of phosphorylation of 4.1R and adducin by PKC on membrane mechanical function. We identified Ser-312 in the 16-kDa domain of 4.1R as the PKC phosphorylation using digestion and Using antibodies raised against phosphopeptides of 4.1R and adducin, we documented significant differences in the time course of the phosphorylation of adducin and 4.1R by PKC in intact Although adducin was phosphorylated rapidly by the activation of atypical PKC as there was a significant delay in the phosphorylation of 4.1R because of the delayed recruitment of conventional PKC as to the membrane. This differential time course in the phosphorylation of 4.1R and adducin in conjunction with membrane mechanical stability measurements enabled us to document that, although the phosphorylation of adducin by PKC has little effect on membrane mechanical stability, the phosphorylation of 4.1R results in a marked decrease in membrane mechanical stability. We further showed that phosphorylated 4.1R purified from intact membranes a decreased ability to form a ternary complex with spectrin and actin and to interact with red cell membrane proteins as The findings from the present enabled us to define a regulatory role for 4.1R phosphorylation in the dynamic regulation of red cell membrane properties. PMA, and from and from and antibodies from The domain of 4.1R was as Y. Takakuwa Y. H. T. S. 1995; PubMed Scopus Google Scholar). was purified from as (8Manno S. Takakuwa Y. Nagao K. Mohandas N. J. Biol. Chem. 1995; 270: 5659-5665Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), and actin was from In of with from of blood was from in a blood of as an The using an of and with The in the The with a of or A in for and with or for of as of of the of 4.1R phosphorylated in intact membranes by from with or for by and the 4.1R band was and from the in The 4.1R was to digestion with on and by The was for in by on to membranes and with an or for phosphorylated binding sites by the membrane with for in Tris-buffered The was with in and for The was with and for with The was with with and to to the A membrane was for in by and in of the phosphorylated The band was by of the of of was from membranes of with the was from and purified by a with in The was with in and was from the with a of in A. was by and using an The was and against The of was using the on the of from and in and the using and was used as the phosphorylated protein of for of 4.1R and that 4.1R phosphorylated Ser-312 was we the phosphorylated to of the 16-kDa domain of 4.1R a The was an N-terminal to raised in of in an of from the was from blood and to for The adducin was in using the to of human or of human R. Bennett V. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar). The in is and of membrane mechanical stability, from with or in the of A for or The in and by the as N. Mohandas N. Scholar, N. M. Rossi M. Shohet S.B. Blood. PubMed Google Scholar). to a of and in the deformability as a function of time and membrane the decreases with The of in is a of membrane mechanical stability, and the time for the to is a quantitative of decreased stability. for membranes with various of 4.1R and adducin phosphorylation of from of 4.1R was from of with or the with A with or for from these red cells in and for The cell for and the 4.1R with the membranes and the 4.1R in the by with both an that phosphorylated 4.1R and an that both phosphorylated and of P-4.1R, and GPC of from with for or of cell was to of by for The was for and the cell was using The by using antibodies against 4.1R, P-4.1R, and of the of 4.1R and to a with and ability of 4.1R and to form a ternary complex with spectrin and actin was using a with and purified spectrin from as (25Correas I. Leto T.L. Speicher D.W. Marchesi V.T. J. Biol. Chem. 1986; 261: 3310-3315Abstract Full Text PDF PubMed Google Scholar). and spectrin with phosphorylated or 4.1R in binding for on to to a of in binding and for of the and of the proteins for their by protein during the of 4.1R, P-4.1R, and spectrin was using the Biochem. PubMed Scopus Google Scholar). of of 4.1R in the domain of 4.1R phosphorylated in intact with PMA, an of in the of a The was by and by by to a and phosphorylated identified by shown in the 16-kDa chymotryptic of 4.1R was phosphorylated by the chymotryptic fragments to the 30-kDa FERM domain, 10-kDa domain, and the C-terminal 22/24-kDa not The identified C. J. H. J. J. S. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) for phosphorylation by are in the 16-kDa domain, and on the identified of the in the 16-kDa domain can be which of the phosphorylated in intact we used was the in the 16-kDa shown in the of from the 16-kDa domain of 4.1R decreased with on showed a decrease in the of various for The of Ser-312 of was decreased that Ser-312 was further was purified from from Although 4.1R be from from normal of the was from of was further purified from 4.1R by with a of in A. Although 4.1R the of was to not This that is 4.1R and that phosphorylation a in showed that the of these was of of that the PKC in 4.1R in that the phosphorylation of 4.1R in intact membranes was was using Although the a band to 4.1R in membranes from with PMA, not 4.1R in membranes with the phorbol This our that Ser-312 is the in 4.1R that is phosphorylated in vivo by PKC. We also an against using the with 4.1R, phosphorylated and not adducin of and in phosphorylation of 4.1R and adducin in intact membranes using the antibodies is shown in 4.1R and adducin a in the of although phosphorylation of adducin the of PMA, there was a the phosphorylation of 4.1R be documented The the of 4.1R and adducin phosphorylation by PKC that different PKC be responsible for the phosphorylation of these skeletal PKC is a of distinct and not are The various in their and the differences in the time course of phosphorylation was by different PKC we for the membrane association of as and as shown in as to be membrane although there is a significant delay in the recruitment of as to the membrane. the differences the of phosphorylation of 4.1R and adducin in membranes be for by the different PKC responsible for the phosphorylation of these skeletal as for adducin and as for the consequences of phosphorylation of 4.1R adducin on membrane mechanical we the membrane mechanical stability of from with for different In for in which adducin was phosphorylated significant in membrane mechanical stability be documented In for both 4.1R and adducin phosphorylated and the membrane mechanical stability was decreased These findings that although phosphorylation of adducin by PKC has effect on membrane mechanical stability, additional phosphorylation of 4.1R decreases the mechanical function of the membrane. in the of 4.1R and adducin phosphorylation and in membrane mechanical stability explored with and antibodies showed that the phosphorylation of adducin that of 4.1R with a of membrane mechanical stability, showed a a decrease in membrane mechanical stability, The membrane mechanical stability not significant further decreases of these findings showed an the PKC phosphorylation of 4.1R and membrane mechanical stability of 4.1R to by effect of phosphorylation on the ability of 4.1R to membrane proteins was by the of 4.1R and from 4.1R was with from red cells and 4.1R be in the In marked was from or 4.1R was with was in the with for phosphorylated 4.1R that although 4.1R was in red cells was phosphorylated in cells the 4.1R from was the phosphorylated form These findings that the phosphorylation of 4.1R the of its with membrane GPC, and band 3. further of the effect of 4.1R phosphorylation on its ability to interact with GPC, the of GPC in from with and was the of by using antibodies against 4.1R, P-4.1R, and GPC with 4.1R that both and phosphorylated 4.1R showed that an of 4.1R was with spectrin and actin in GPC with from membranes and membranes for with in which 4.1R was In marked little GPC was with 4.1R was phosphorylated a with These results that the of 4.1R with GPC is phosphorylation of of 4.1R with and by effect of phosphorylation on the ability of 4.1R to form a ternary complex with spectrin and actin was using a shown in although spectrin be in association with the the of 4.1R to a of spectrin and actin the of spectrin and the ability of to promote spectrin-actin was decreased with 4.1R These findings that the phosphorylation of 4.1R its ability to form a ternary complex with spectrin and and functional studies have documented an important role for the spectrin-based membrane skeleton in regulating membrane mechanical function (1Mohandas N. Chasis J.A. Semin. Hematol. 1993; 30: 171-192PubMed Google Scholar). decreased self-association of spectrin from in either β-spectrin and the of spectrin-actin-4.1R by in β-spectrin and 4.1R have been shown to decrease membrane mechanical stability (1Mohandas N. Chasis J.A. Semin. Hematol. 1993; 30: 171-192PubMed Google Scholar, 5Takakuwa Y. Tchernia G. Rossi M. Benabadji M. Mohandas N. J. Clin. Investig. 1986; 78: 80-85Crossref PubMed Scopus (87) Google Scholar). β-spectrin, 4.1R, adducin, and dematin can be phosphorylated by a number of different kinases and dephosphorylated by various phosphatases C.M. Gascard P. Semin. Hematol. 1992; 29: 244-292PubMed Google Scholar). In vitro studies using purified proteins have shown that phosphorylation can alter skeletal protein function (6Boivin P. Biochem. J. 1988; 256: 689-695Crossref PubMed Scopus (58) Google Scholar, 7Cohen C.M. Gascard P. Semin. Hematol. 1992; 29: 244-292PubMed Google Scholar). However, our understanding of how phosphorylation of various red cell membrane skeletal proteins can mechanical function of intact red cell membranes has not been well defined. A of the present is the that phosphorylation of 4.1R, not adducin, by PKC membrane mechanical function. A of the present In to studies that on the of to we used antibodies that the phosphorylated of the The of these antibodies enabled us to document significant differences in time course of phosphorylation of adducin and 4.1R in intact The phosphorylation of adducin is the result of activation of as by stimulation, the phosphorylation of 4.1R is by the delayed recruitment of as to the membrane. This differential time course the phosphorylation of 4.1R and adducin in conjunction with membrane mechanical stability measurements enabled us to that although the phosphorylation of adducin by PKC has little effect on membrane mechanical stability, the additional phosphorylation of 4.1R results in a marked decrease in membrane mechanical stability. is for membrane mechanical stability. In solution, both 4.1R and adducin spectrin-actin However, phosphorylation of either 4.1R or adducin by PKC decreases its ability to promote spectrin-actin in vitro (30Ling E. Danilov Y.N. Cohen C.M. J. Biol. Chem. 1988; 263: 2209-2216Abstract Full Text PDF PubMed Google Scholar). is to that in intact membranes PKC phosphorylation of adducin little effect on membrane mechanical phosphorylation of both 4.1R and adducin in decreased membrane mechanical This that 4.1R phosphorylation in intact membranes spectrin-actin to decreased membrane mechanical stability. be that we not the that 4.1R and adducin in regulating membrane mechanical function. Although a number of different in purified 4.1R are phosphorylated by PKC in solution, Ser-312 in the 16-kDa domain of 4.1R is phosphorylated in intact studies showed that 4.1R phosphorylated Ser-312 a decreased ability to promote spectrin-actin that the 4.1R and the N-terminal domain of β-spectrin and actin filaments is by the 10-kDa domain that the phosphorylation of Ser-312 a in the domain of phosphorylated 4.1R also decreased for with band 3 and GPC, by its 30-kDa FERM domain, a in domain as Indeed, a in 4.1R on phosphorylation can be from our of differences in the of and 4.1R by from these findings that the 16-kDa domain plays a regulatory role in the of 4.1R by phosphorylation and of Ser-312 by PKC and protein In to our a previous S.H. Low P.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) to document in membrane mechanical stability phosphorylation of 4.1R by PKC. The for the is the result of of 4.1R during the and of in the S.H. Low P.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In we that 4.1R is dephosphorylated during of the in the of the A. A that for our findings is shown in In the 4.1R with with the domain of GPC its 30-kDa FERM domain and with spectrin and actin its 10-kDa of Ser-312 in the 16-kDa domain decreases the of both to GPC and to spectrin and The decreased of the by phosphorylation to decreased membrane as by decreased membrane mechanical stability. In to the present an (8Manno S. Takakuwa Y. Nagao K. Mohandas N. J. Biol. Chem. 1995; 270: 5659-5665Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) documented that the phosphorylation of β-spectrin by casein kinase I also decreases membrane mechanical stability. these findings a regulatory role for the phosphorylation of skeletal proteins in the dynamic regulation of membrane properties.