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Brain phosphoprotein phosphatase activities in Alzheimer disease
THIRD INTERNATIONAL
CONFERENCE
ON ALZHEIMER’S
DISEASE
210
212
PROTEIN KINASE C PHOSPHORYLATES THE TUBULINBINDING DOMAIN OF TAU PROTEIN. *I. Correas, J. Diaz-Nido and J. Avila. Centro de Biologia Molecular. Authoma de Madrid. E-28049 Facultad de Ciencias. Universidad Madrid. Spain.
PHOSPHORYLATION OF TAU PROTEIN BY CAMP-DEPENDENT PROTEIN KINASE: IDENTIFICATION OF PHOSPHORYLATION SITES AND EFFECT ON TAU FUNCTION, Clay W Scott, Russell C. Spreen, Joseph L. Herman, Francis H. Chow, and Claudia B. Caputo, ICI Pharmaceuticals Group, ICI Americas, Wilmington, DE 19897. USA. Neurofibtillary tangles are neuropathologic lesions that occur in Alzheimer’s disease. Abnormally phosphorylated tau protein is an integral component of paired helical filaments (PHF), the structural unit within neurofibrillary tangles. The actual sites of phosphorylation on normal and PHF-tau have not been mapped, and the effect of abnormal phosphorylation on tau function has not been described. We have begun to map the sites on human recombinant tau that are. phosphorylated in vitro using various purified protein kinases, and have examined the effect of these phosphotylations on tau function. CAMP-dependent protein kinase (PKA) phosphorylated the shortest tau isoform (T3) to a stoichiometry approaching 2 mol phosphate/m01 tau. The phosphorylation sites were identified by digesting phosphorylated tau with proteases. separating the peptides by reversed phase HPLC, and analyzing the isolated peptides by liquidsecondary ion mass spectrometry (L-SIMS). Phosphoamino acid analysis and manual Edman degradation combined with L-SIMS conclusively identified three phosphorylation sites (Ser 156,235 and 267 of T3) and suggested diphosphorylation of a short peptide near the C-terminus. Phosphorylation of tau by PKA caused a decrease in the rate of tau-induced microtubule (MT) assembly, but had no effect on the steady state assembly of MTs. Phosphorylated and nonphosphorylated tau showed similar dosedependent bundling of microtubules, indicating that phosphorylation by PKA did not affect the ability of tau to crosslink adjacent microtubules. These results link the phosphorylation of defined residues on tau with decreased MT assembly-promoting activity.
The stabilization of the neural cytoskeleton by the interaction of tau protein with tubulin may be regulated by tau phosphorylation. Abnormal hyperphosphorylation of tau may abolish its binding to tubulin leading to its deposition into Alzheimer’s disease neurofibrillary tangles. The reduced affinity of phosphorylated tau for tubulin could be accounted for direct phosphorylation on sites of tau tubulin-binding domain and/or induced conformational changes. We have attempted to identify phosphorylation sites at the tubulin-binding domain of tau. In vitro phosphorylation of bovine tau protein with different protein kinases (protein kinase C, CAMPdependent protein kinase, casein kinase II and a proline-directed serine/threonine protein kinase) has been carried out. Limited proteolysrs assays have indicated that these protein kinases phosphorylate tau protein on sites localized in different regions of the molecule and that phosphorylation sites for protein kinase C could be located within the tubulin-binding domain. Direct phosphorylation of synthetic peptides corresponding to the cysteinecontaining tubulin-binding region present in both fetal and adult tau isoforms demonstrate that serine 313 is modified by protein kinase C. Phosphorylation of the synthetic peptide by this protein kinase diminishes its binding to tubulin, as compared with the unphosphorylated peptide. The phosphorylatron of the tubulin-bmding domain of tau protein may constitute a mechanism to weaken the interaction of tau with tubulin, thus destabilizing microtubules and, therefore, modifying the neural cytoskeleton in response to the extracellular signals which activate protein kinase C.
213
211 CYCLICAMP-DEPENDENT THE ELECTROPHORETIC
PROTEIN KINASE INDUCES A SHIFT IN MOBILITY OF HUMAN TAU
D.P. Hanger’, T.L.F. Loviny’, J. Robertson’, K.J. Murray’, and B _H. Anderton' .
M. Goedert’.
‘Department of Neu mscience, Institute of Psychiatry, London, UK, ‘Laboratory of Molecular Biology, MRC, Cambridge, UK, %mithKline Beecham, The Frythe, Welwyn, Herts, UK. T~U is an integral component of the paired helical filaments which make up the characteristic neurofibrilIary tangles in Alzheimer’s disease (AD). Control human brain contains a set of six tau isoformo which can separated by SDS-PAGE whereas brain affected by Alsheimer pathology shows a different pattern of tau isOfOrmS (PHF-tau) due to hyperphcsphorylation. Recombinant human tau (R-tau) and tau extracted from control human brain (C-tau) were phosphorylated in vitro by cyclic AMPdependent protein kinass (PKA) . Short incubatior~s with PKA lead to incorporation of phosphate into the protein without causing any apparent alteration in the electrophoretic mobility. Longer incubation of each R-tau isoform with PKA produced two further phosphorylated tau species exhibiting apparent M. increases of approximately 1.5 and 4 kDa compared to the parent isoform on SDS-PAGE. Two fragments of one of the R-tau isoforms, from which either the N- or the C-terminus was missing, were incubated with PKA and these proteins also displayed small reductions in electrophoretic mobility on SDS-PAGE although the magnitude of the shift was smaller than with the complete tau isoform. Two mutant forms of one of the R-tau proteins, containing an alanine substitution at either serinelg6 (a site phosphorylated in PHF-tau) or serir~?“~ (identified as the site of in vitro phosphorylation by caIcium/calmodulin-dependent protein kinase II) were also phosphorylated by PKA in a manner which reduced their mobility on SDS-PAGE by about the same extent as that of the non-mutated isoform. C-tau was also phosphorylated by PKA and the electrophoretic mobility of at least some of the tau isoforms was reduced. Western blots of the phosphorylated tau species produced by PKA from R-tau and C-tau did not align exactly with PHF-tau. We conclude that phosphorylation of tau by PKA may contribute to the formation of PHF-tau in Wheimer’s disease but that it is unlikely to be the only responsible kinase.
BRAIN PHOSPHOPROTEIN PHOSPHATASE ACTIVITIES IN ALZHEIMER DISEASE, C.X. Gong, T.J. Singh, I. Grundke-Iqbal and K Iqbal. New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA Microtubule-associated protein tau has been shown to be abnormally phosphorylated in Alzheimer disease (AD) brain and to be the major component of paired helical filaments. Our previous studies (Proc. Natl. Acad. Sci. USA 83:4913-4917,1986, ibid 8656465650,1989) showing the dephosphorylation of abnormally phosphotylated tau on treatment with alkaline phosphatase jut &Q suggested that the abnormal phosphorylation might be in part the result of a deficiency of the phosphoprotein phosphatase system in the affected neurons in AD. In this study, we have used UP-labeled phosphorylase kinase as substrate to measure phosphoprotein phosphatases in AD and control brains. Four types of phosphoprotein phosphatase activities (type 1, type 2A, type 2B and type 2C) of both frontal grey and white matters of five AD brains were determined and compared with those of four age-matched control brains. Phosphatase 2C activity in grey matter was statistically significantly lower in AD brain than in the control group. These results suggest that the abnormal phosphorylation of tau in AD brain could result from a protein dephosphorylation defect in viva. The nature and the mechanism(s) of the decreased phosphoprotein phosphatase activities are still to be clarified. However, these findings suggest a possible way to understand the cause of abnormal phosphorylation and, consequently, the pathogenesis of AD. (Supported in part by NIH grants AG08076, AG05892, NS18105, and AGO4220 and a grant from the American Health Assistance Foundation, Rockville, MD).
214 ISOLATED ALZHEIMER’S SEQUENCE RECOGNIZED ANTIBODIES.
PHF AND PHF-TAU (AaS) POLYPEPTIDES 1 N DISEASE CONTAlN EPITOPES FROM TEE WHOLE DIFFERENT TAU ISOFORMS AND MODIFIED SITES BY RT97 AND SD8 ANTI-NEUROFILAMENT J.P.Brioal.A.M. Couckt. B.H. Anderton2. tlaborat. of F’athology and
OF
Electron Micmsmpy. Universit& L.ibre de Bruxelles. Belgium and 2Depm. of Neuroscience. Insititute of Psy&iatry. London. UK
CONFERENCE
ON ALZHEIMER’S
DISEASE
210
212
PROTEIN KINASE C PHOSPHORYLATES THE TUBULINBINDING DOMAIN OF TAU PROTEIN. *I. Correas, J. Diaz-Nido and J. Avila. Centro de Biologia Molecular. Authoma de Madrid. E-28049 Facultad de Ciencias. Universidad Madrid. Spain.
PHOSPHORYLATION OF TAU PROTEIN BY CAMP-DEPENDENT PROTEIN KINASE: IDENTIFICATION OF PHOSPHORYLATION SITES AND EFFECT ON TAU FUNCTION, Clay W Scott, Russell C. Spreen, Joseph L. Herman, Francis H. Chow, and Claudia B. Caputo, ICI Pharmaceuticals Group, ICI Americas, Wilmington, DE 19897. USA. Neurofibtillary tangles are neuropathologic lesions that occur in Alzheimer’s disease. Abnormally phosphorylated tau protein is an integral component of paired helical filaments (PHF), the structural unit within neurofibrillary tangles. The actual sites of phosphorylation on normal and PHF-tau have not been mapped, and the effect of abnormal phosphorylation on tau function has not been described. We have begun to map the sites on human recombinant tau that are. phosphorylated in vitro using various purified protein kinases, and have examined the effect of these phosphotylations on tau function. CAMP-dependent protein kinase (PKA) phosphorylated the shortest tau isoform (T3) to a stoichiometry approaching 2 mol phosphate/m01 tau. The phosphorylation sites were identified by digesting phosphorylated tau with proteases. separating the peptides by reversed phase HPLC, and analyzing the isolated peptides by liquidsecondary ion mass spectrometry (L-SIMS). Phosphoamino acid analysis and manual Edman degradation combined with L-SIMS conclusively identified three phosphorylation sites (Ser 156,235 and 267 of T3) and suggested diphosphorylation of a short peptide near the C-terminus. Phosphorylation of tau by PKA caused a decrease in the rate of tau-induced microtubule (MT) assembly, but had no effect on the steady state assembly of MTs. Phosphorylated and nonphosphorylated tau showed similar dosedependent bundling of microtubules, indicating that phosphorylation by PKA did not affect the ability of tau to crosslink adjacent microtubules. These results link the phosphorylation of defined residues on tau with decreased MT assembly-promoting activity.
The stabilization of the neural cytoskeleton by the interaction of tau protein with tubulin may be regulated by tau phosphorylation. Abnormal hyperphosphorylation of tau may abolish its binding to tubulin leading to its deposition into Alzheimer’s disease neurofibrillary tangles. The reduced affinity of phosphorylated tau for tubulin could be accounted for direct phosphorylation on sites of tau tubulin-binding domain and/or induced conformational changes. We have attempted to identify phosphorylation sites at the tubulin-binding domain of tau. In vitro phosphorylation of bovine tau protein with different protein kinases (protein kinase C, CAMPdependent protein kinase, casein kinase II and a proline-directed serine/threonine protein kinase) has been carried out. Limited proteolysrs assays have indicated that these protein kinases phosphorylate tau protein on sites localized in different regions of the molecule and that phosphorylation sites for protein kinase C could be located within the tubulin-binding domain. Direct phosphorylation of synthetic peptides corresponding to the cysteinecontaining tubulin-binding region present in both fetal and adult tau isoforms demonstrate that serine 313 is modified by protein kinase C. Phosphorylation of the synthetic peptide by this protein kinase diminishes its binding to tubulin, as compared with the unphosphorylated peptide. The phosphorylatron of the tubulin-bmding domain of tau protein may constitute a mechanism to weaken the interaction of tau with tubulin, thus destabilizing microtubules and, therefore, modifying the neural cytoskeleton in response to the extracellular signals which activate protein kinase C.
213
211 CYCLICAMP-DEPENDENT THE ELECTROPHORETIC
PROTEIN KINASE INDUCES A SHIFT IN MOBILITY OF HUMAN TAU
D.P. Hanger’, T.L.F. Loviny’, J. Robertson’, K.J. Murray’, and B _H. Anderton' .
M. Goedert’.
‘Department of Neu mscience, Institute of Psychiatry, London, UK, ‘Laboratory of Molecular Biology, MRC, Cambridge, UK, %mithKline Beecham, The Frythe, Welwyn, Herts, UK. T~U is an integral component of the paired helical filaments which make up the characteristic neurofibrilIary tangles in Alzheimer’s disease (AD). Control human brain contains a set of six tau isoformo which can separated by SDS-PAGE whereas brain affected by Alsheimer pathology shows a different pattern of tau isOfOrmS (PHF-tau) due to hyperphcsphorylation. Recombinant human tau (R-tau) and tau extracted from control human brain (C-tau) were phosphorylated in vitro by cyclic AMPdependent protein kinass (PKA) . Short incubatior~s with PKA lead to incorporation of phosphate into the protein without causing any apparent alteration in the electrophoretic mobility. Longer incubation of each R-tau isoform with PKA produced two further phosphorylated tau species exhibiting apparent M. increases of approximately 1.5 and 4 kDa compared to the parent isoform on SDS-PAGE. Two fragments of one of the R-tau isoforms, from which either the N- or the C-terminus was missing, were incubated with PKA and these proteins also displayed small reductions in electrophoretic mobility on SDS-PAGE although the magnitude of the shift was smaller than with the complete tau isoform. Two mutant forms of one of the R-tau proteins, containing an alanine substitution at either serinelg6 (a site phosphorylated in PHF-tau) or serir~?“~ (identified as the site of in vitro phosphorylation by caIcium/calmodulin-dependent protein kinase II) were also phosphorylated by PKA in a manner which reduced their mobility on SDS-PAGE by about the same extent as that of the non-mutated isoform. C-tau was also phosphorylated by PKA and the electrophoretic mobility of at least some of the tau isoforms was reduced. Western blots of the phosphorylated tau species produced by PKA from R-tau and C-tau did not align exactly with PHF-tau. We conclude that phosphorylation of tau by PKA may contribute to the formation of PHF-tau in Wheimer’s disease but that it is unlikely to be the only responsible kinase.
BRAIN PHOSPHOPROTEIN PHOSPHATASE ACTIVITIES IN ALZHEIMER DISEASE, C.X. Gong, T.J. Singh, I. Grundke-Iqbal and K Iqbal. New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA Microtubule-associated protein tau has been shown to be abnormally phosphorylated in Alzheimer disease (AD) brain and to be the major component of paired helical filaments. Our previous studies (Proc. Natl. Acad. Sci. USA 83:4913-4917,1986, ibid 8656465650,1989) showing the dephosphorylation of abnormally phosphotylated tau on treatment with alkaline phosphatase jut &Q suggested that the abnormal phosphorylation might be in part the result of a deficiency of the phosphoprotein phosphatase system in the affected neurons in AD. In this study, we have used UP-labeled phosphorylase kinase as substrate to measure phosphoprotein phosphatases in AD and control brains. Four types of phosphoprotein phosphatase activities (type 1, type 2A, type 2B and type 2C) of both frontal grey and white matters of five AD brains were determined and compared with those of four age-matched control brains. Phosphatase 2C activity in grey matter was statistically significantly lower in AD brain than in the control group. These results suggest that the abnormal phosphorylation of tau in AD brain could result from a protein dephosphorylation defect in viva. The nature and the mechanism(s) of the decreased phosphoprotein phosphatase activities are still to be clarified. However, these findings suggest a possible way to understand the cause of abnormal phosphorylation and, consequently, the pathogenesis of AD. (Supported in part by NIH grants AG08076, AG05892, NS18105, and AGO4220 and a grant from the American Health Assistance Foundation, Rockville, MD).
214 ISOLATED ALZHEIMER’S SEQUENCE RECOGNIZED ANTIBODIES.
PHF AND PHF-TAU (AaS) POLYPEPTIDES 1 N DISEASE CONTAlN EPITOPES FROM TEE WHOLE DIFFERENT TAU ISOFORMS AND MODIFIED SITES BY RT97 AND SD8 ANTI-NEUROFILAMENT J.P.Brioal.A.M. Couckt. B.H. Anderton2. tlaborat. of F’athology and
OF
Electron Micmsmpy. Universit& L.ibre de Bruxelles. Belgium and 2Depm. of Neuroscience. Insititute of Psy&iatry. London. UK