Biomedical subjects

S D Yang

Publications and source records attributed to S D Yang.

At least 55 records · Page 3Linked to original sources

PubMed1994

Identification of -R-X-(X)-S/T-X3-S/T- as consensus sequence motif for autophosphorylation-dependent protein kinase.

To identify consensus sequence motif for a new family of protein kinase termed autophosphorylation-dependent protein serine/threonine kinase (auto-kinase), we have tested several synthetic peptides. The well established protein serine/threonine kinases such as cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase (CaM-kinase), and protein kinase C were found to be inactive toward phosphorylation of syntide-3 (RPRPASVPPSPSLSRHA), which turned out to be an excellent substrate only for auto-kinase, indicating that syntide-3 is a specific substrate for auto-kinase. Modification of syntide-3 to become RPRPASVPPS/T did not affect the activity of auto-kinase. By contrast, autokinase became rather or almost inactive when the peptide was modified to become RPRPASVPPA/G/F/K/R/D/E/Y, indicating that amino acid number 10 in syntide-3 is crucial to the sequence motif recognized by auto-kinase. Phosphorylation of myelin basic protein (MBP) by autokinase revealed that auto-kinase predominantly phosphorylates MBP on one particular site with RT-T(p)HYGS as the phosphorylation site sequence, which could not be phosphorylated by any other reported MBP kinases including cAMP-dependent protein kinase, CaM-kinase, protein kinase C, mitogen-activated protein kinase, and kinase FA/GSK-3. Taken together, the results provide initial evidence that -Arg-X-(X)-Ser/Thr-X3-Ser/Thr- may represent a unique consensus sequence motif specifically recognized by autophosphorylation-dependent protein kinase, a new family of multi-substrate/multifunctional protein serine/threonine kinase.

Amino Acid Sequence↗

PubMed1994

Okadaic acid, a serine/threonine phosphatase inhibitor, induces tyrosine dephosphorylation/inactivation of protein kinase FA/GSK-3 alpha in A431 cells.

Alkaloids↗

PubMed1994

Tyrosine dephosphorylation and concurrent inactivation of protein kinase FA/GSK-3 alpha by genistein in A431 cells.

Modulation of protein kinase FA/GSK-3 alpha by tyrosine phosphorylation in A431 cells was investigated. Kinase FA/GSK-3 alpha was found to exist in a highly tyrosine-phosphorylated/activated state in resting cells but could become tyrosine-dephosphorylated and inactivated down to less than 30% of control values in a concentration-dependent manner by 50-400 microM genistein (a specific tyrosine kinase inhibitor), as demonstrated by metabolic 32P-labeling of the cells followed by immunoprecipitation and two-dimensional phosphoamino acid analysis and by immunodetection in an antikinase FA/GSK-3 alpha immunoprecipitate kinase assay. Taken together, the results provide evidence that kinase FA/GSK-3 alpha may exist in a highly tyrosine-phosphorylated/activated state in resting cells which can be tyrosine-dephosphorylated and inactivated by extracellular stimulus and that tyrosine kinase(s) and/or tyrosine phosphatase(s) may play a role in the modulation of kinase FA/GSK-3 alpha activity in cells.

Amino Acid Sequence↗

PubMed1994

Tumor promoter phorbol ester reversibly modulates tyrosine dephosphorylation/inactivation of protein kinase FA/GSK-3 alpha in A431 cells.

The signal transduction mechanism of protein kinase FA/GSK-3 alpha by tyrosine phosphorylation in A431 cells was investigated. Kinase FA/GSK-3 alpha was found to exist in a highly tyrosine-phosphorylated/activated state in resting cells but could be tyrosine-dephosphorylated and inactivated to approximately 60% of the control level when cells were acutely treated with 1 microM tumor promoter phorbol ester (TPA) at 37 degrees C for 30 min, as demonstrated by metabolic 32P-labeling the cells, followed by immunoprecipitation and two-dimensional phosphoamino acid analysis and by immunodetection in an antikinase FA/GSK-3 alpha immunoprecipitate kinase assay. Conversely, when cells were chronically treated with 1 microM TPA at 37 degrees C for 24 h and processed under identical conditions, kinase FA/GSK-3 alpha was found to be rephosphorylated on tyrosine residue and reactivated to approximately 130% of the original control level. Taken together, the results provide initial evidence that the phosphotyrosine content and cellular activity of kinase FA/GSK-3 alpha can be modulated in a reversible manner by short-term and long-term exposure of A431 cells to TPA. Since acute exposure of cells to TPA causes up-regulation of cellular protein kinase C (PKC) activity and prolonged exposure to TPA causes down-regulation of PKC, the results further suggest that the TPA-mediated modulation of PKC may play a role in the regulation of tyrosine phosphorylation and concurrent activation of kinase FA/GSK-3 alpha in cells, representing a new mode of signal transduction pathway for the regulation of this multisubstrate/multifunctional protein kinase in cells.

Amino Acid Sequence↗

PubMed1994

Autophosphorylation-dependent protein kinase predominantly phosphorylates Ser115, the in vivo site in brain myelin basic protein.

In a previous report [Yang et al., (1987a), J. Biol Chem. 262, 7034-7040], a cyclic-AMP- and calcium-independent brain kinase which requires autophosphorylation for activity was identified as a very potent myelin basic protein (MBP) kinase. In this report, the phosphorylation sites of MBP by this autophosphorylation-dependent protein kinase (autokinase) are further determined by two-dimensional electrophoresis/thin-layer chromatography, phosphoamino acid analysis, high-performance liquid chromatography, tryptic peptide mapping, sequential manual Edman degradation, and direct peptide sequencing. Autokinase phosphorylates MBP on both threonine and serine residues. Three major tryptic phosphopeptide peaks were resolved by C18-reversed phase high-performance liquid chromatography. Sequential manual Edman degradation together with direct sequence analysis revealed that FS(p)WGAEGQKPGFGYGGR is the phosphorylation site sequence (molar ratio approximately 1.0) for the first major phosphopeptide peak. When mapping with bovine brain MBP sequence, we finally demonstrate Ser115, one of the in vivo phosphorylation sites in MBP, as the major site phosphorylated by autokinase, implicating a physiologically relevant role of autokinase in the regulation of brain myelin function. By using the same approach, we also identified HRDT(p)GILDSLGR (molar ratio approximately 0.9) and TT(p)HYGSLPQK (molar ratio approximately 0.8) as the major phosphorylation site sequences in 32P-MBP phosphorylated by autokinase, further indicating that -Arg-X-Ser/Thr-(neutral amino acid)3-(amino acid-containing hydroxyl group such as Ser/Glu/Asp)-(neutral amino acid)2-may represent a unique consensus sequence motif specifically recognized by this autophosphorylation-dependent multisubstrate/multifunctional protein kinase in the brain.

Amino Acid Sequence↗

PubMed1994

Autophosphorylation-dependent protein kinase phosphorylates Ser25, Ser38, Ser65, Ser71, and Ser411 in vimentin and thereby inhibits cytoskeletal intermediate filament assembly.

The autophosphorylation-dependent protein kinase has been identified as a potent vimentin kinase that incorporates 2 mol of phosphates per mol of protein and generates five major phosphorylation sites in vimentin. Tryptic phosphopeptide mapping by high-performance liquid chromatography followed by sequential manual Edman degradation and direct peptide sequence analysis revealed that Ser-25, Ser-38, Ser-65, and Ser-71 in the amino-terminal domain and Ser-411 in the carboxyl-terminal domain are the phosphorylation sites in vimentin phosphorylated by this kinase, indicating that autophosphorylation-dependent protein kinase is a potent and unique vimentin kinase. Functional study further revealed that phosphorylation of vimentin by autophosphorylation-dependent protein kinase can completely inhibit polymerization and assembly of the cytoskeletal intermediate filament as demonstrated by electron microscopic analysis. Taken together, the results provide initial evidence that the autophosphorylation-dependent protein kinase may function as a vimentin kinase involved in the structure-function regulation of the cytoskeletal system. The results also support the notion that this cyclic nucleotide- and calcium-independent protein kinase may function as a multisubstrate/multifunctional protein kinase involved in the regulation of diverse cell functions.

Amino Acid Sequence↗

PubMed1994

Protein kinase FA/glycogen synthase kinase-3 predominantly phosphorylates the in vivo site Thr97-Pro in brain myelin basic protein: evidence for Thr-Pro and Ser-Arg-X-X-Ser as consensus sequence motifs.

In a previous study, protein kinase FA/glycogen synthase kinase-3 (FA/GSK-3) was identified as a myelin basic protein (MBP) kinase associated with intact brain myelin. In this report, the phosphorylation sites of MBP by kinase FA/GSK-3 were further determined by two-dimensional electrophoresis/TLC, phosphoamino acid analysis, tryptic peptide mapping, Edman degradation, and direct sequencing. Kinase FA/GSK-3 phosphorylates MBP on both threonine and serine residues. Three tryptic phosphopeptide peaks were resolved by C18 reverse-phase HPLC. Sequential manual Edman degradation together with direct sequence analysis revealed that T(p)PPPSQGK is the phosphorylation site sequence for the first major phosphopeptide peak. When mapping with the bovine brain MBP sequence, we finally demonstrate Thr97-Pro, one of the in vivo phosphorylation sites in MBP, as the major site phosphorylated by kinase FA/GSK-3, implicating a physiologically relevant role of FA/GSK-3 in the regulation of brain myelin function. By using the same approach, we also identified NIVT94(p)PR as the phosphorylation site sequence in the second major tryptic phosphopeptide derived from [32P]MBP phosphorylated by kinase FA/GSK-3, further indicating that kinase FA/GSK-3 represents a Thr-Pro motif-directed MBP kinase involved in the phosphorylation of brain myelin.

Amino Acid Sequence↗

PubMed1994

Protein kinase FA/glycogen synthase kinase-3 alpha after heparin potentiation phosphorylates tau on sites abnormally phosphorylated in Alzheimer's disease brain.

Previously, we identified protein kinase FA/glycogen synthase kinase-3 alpha (GSK-3 alpha) as a brain microtubule-associated tau kinase that phosphorylates Ser235 and Ser404 of tau and causes its electrophoretic mobility shift in gels, a unique property characteristic of paired helical filament-associated pathological tau (PHF-tau) in Alzheimer's disease brains. In this study, we found that the activity of kinase FA/GSK-3 alpha towards phosphorylation of brain tau could be stimulated approximately fourfold by heparin. The phosphorylation molar ratio was increased simultaneously up to 9 mol of phosphates/mol of tau, resulting in a reduced mobility of tau with an apparent molecular mass shift to approximately 68 kDa in sodium dodecyl sulfate gels, which is very similar to that observed in Alzheimer-tau. Tryptic digestion of 32P-labelled tau, followed by HPLC and two-dimensional separation on TLC cellulose plates, revealed eight major phosphopeptides. Phosphoamino acid analysis together with sequential manual Edman degradation and protein sequence analysis further revealed that, in addition to Ser235 and Ser404, heparin generated Thr212, Thr231, Ser262, Ser324, and Ser356, the five extra phosphorylation sites in tau. As Ser235, Ser262, Ser324, Ser356, and Ser404 (particularly the site of Ser262) have been identified as five of the most potent sites in tau responsible for reducing microtubule binding possibly involved in neuronal degeneration, and Thr231, Ser235, Ser262, and Ser404 are four of the most well documented sites abnormally phosphorylated in Alzheimer-tau, the results provide initial evidence that protein kinase FA/GSK-3 alpha after heparin potentiation may represent one of the most potent systems possibly involved in the abnormal phosphorylation of PHF-tau in Alzheimer's disease brains.

Alzheimer Disease↗

PubMed1994

[Pharmacognosy of Ranunculus chinensis Bge].

Ranunculus chinensis is a folk herb popular in some districts of Yunnan Province. The paper deals with the plant resources, macroscopic shape and microscopic characteristics of this herb so as to provide referential information for clinics, research institutes and herbal market.

Drugs, Chinese Herbal↗

PubMed1993

Synergistic control mechanism for abnormal site phosphorylation of Alzheimer's diseased brain tau by kinase FA/GSK-3 alpha.

When a synthetic peptide fragment (VAVVRTPPKSPSSAK) which corresponds to amino acid residues 226-240 from brain microtubule-associated protein tau was used as a testing substrate, we found that protein kinase FA/GSK-3 alpha was almost inactive towards this substrate. In sharp contrast, when Ser-10 of this peptide was replaced by a phosphoserine, the phosphopeptide fragment (VAVVRTPPKS(p)PSSAK) became an excellent substrate for kinase FA/GSK-3 alpha. Sequential manual Edman degradation together with phosphoamino acid analysis and protein sequencing further revealed that Thr-6 of the peptide fragment which corresponds to an important abnormal phosphorylation site Thr-231 in Alzheimer's diseased brain tau was the only site that was greatly phosphorylated, demonstrating that a pre-phosphorylation becomes a prerequisite and is essential for promoting phosphorylation of Thr-231. Taken together, the results provide initial evidence that kinase FA/GSK-3 alpha mediates a synergistic phosphorylation control mechanism involved in the abnormal site phosphorylation of Alzheimer's diseased brain tau.

Alzheimer Disease↗

PubMed1993

Cyclic modulation of cytoskeleton assembly-disassembly by the ATP.Mg-dependent protein phosphatase activator (kinase FA).

The ATP.Mg-dependent protein phosphatase activating factor (FA) has been identified as a microtubule protein kinase and as a microtubule protein phosphatase activator. FA could phosphorylate microtubule-associated tau protein up to 4 moles of phosphates per mole of protein. However, more than 80% of the phosphates in 32P-tau phosphorylated by FA could be removed by ATP.Mg-dependent protein phosphatase and the tau phosphatase activity was FA-dependent. Functional study further revealed that as a tau kinase, FA could phosphorylate tau and thereby inhibits cross-linking copolymerization of tau with tubulin and actin filaments whereas as a tau phosphatase activating factor, FA could promote copolymerization of tau with tubulin and actin filaments. Taken together, the results provide evidence that a cyclic modulation of cytoskeleton assembly-disassembly can be controlled by FA, representing an efficient cyclic cascade mechanism for rapid structural and functional regulation of cytoskeletal system in the central nervous system.

Adenosine Triphosphate↗

PubMed1993

Immunological and biochemical study on tissue and subcellular distributions of protein kinase FA (an activating factor of ATP.Mg-dependent protein phosphatase): a simplified and efficient procedure for high quantity purification from brain.

Although protein kinase FA/GSK-3 alpha (an activating factor of ATP.Mg-dependent protein phosphatase) has been established as a cytosolic enzyme in mammalian nonnervous tissues involved in the metabolic regulation, immunological and biochemical studies on tissue and subcellular distributions demonstrate that kinase FA/GSK-3 alpha is in fact a membrane-associated enzyme and most abundantly exists in brain particulate membrane fractions depending on the tissue homogenization conditions. For instance, when brain was homogenized in Polytron without 0.32 M sucrose, approximately 40% of the total FA/GSK-3 alpha was found in the cytosol. However, when brain was homogenized in buffer containing 0.32 M sucrose and in a glass homogenizer with Teflon pestle, more than 80% of the total FA/GSK-3 alpha was found associated with the particulate membrane fractions. By manipulating these findings, we have developed a simplified procedure for purification of homogeneous kinase FA/GSK-3 alpha in high recovery and in a substantial amount from brain tissue. The data explain why kinase FA/GSK-3 alpha cannot be isolated in a reasonable amount from most mammalian tissues for the past years. The specific pure antibody that can specifically recognize kinase FA/GSK-3 alpha from crude tissue extracts together with the high quantity purification of the enzyme as presented in this report provides an initial key step for studies on the role of kinase FA/GSK-3 alpha in the regulation of brain functions especially in the brain particulate membrane fractions.

Amino Acid Sequence↗

PubMed1993

Cyclic modulation of cross-linking interactions of microtubule-associated protein-2 with actin and microtubules by protein kinase FA.

The ATP.Mg-dependent type-1 protein phosphatase activating factor (factor FA) was identified as a brain protein kinase that could phosphorylate microtubule-associated protein-2 (MAP-2) and thereby inhibit cross-linking interactions of MAP-2 with actin filaments and microtubules isolated from porcine brain. The phosphorylation sites were found to be equally located on both projection and microtubule-binding domains of MAP-2. Phosphoamino acid analysis revealed that the phosphorylation sites were on both serine and threonine residues, indicating that factor FA is a serine/threonine-specific MAP-2 kinase. Conversely, factor FA was further identified as a MAP-2 phosphatase activator that could promote the dephosphorylation of 32P-MAP-2 phosphorylated by factor FA itself and thereby potentiate cross-linking interactions of MAP-2 with actin and microtubules. Furthermore, the two opposing functions of factor FA can be selectively modulated in a reciprocal manner by pH change. For instance, alkaline pH could stimulate factor FA to work as a MAP-2 kinase but simultaneously block it to work as a MAP-2 phosphatase activator to potentiate the inhibition on the cross-linking interactions of MAP-2 with actin and microtubules. Taken together, the results provide initial evidence that a cyclic modulation of cross-linking interactions of MAP-2 with actin filaments and microtubules can be controlled by factor FA, representing an efficient cyclic cascade control mechanism for rapid structural and functional regulation of neuronal cytoskeletal system.

Actins↗

PubMed1993

Protein kinase FA/GSK-3 phosphorylates tau on Ser235-Pro and Ser404-Pro that are abnormally phosphorylated in Alzheimer's disease brain.

Previously, we identified protein kinase FA/glycogen synthase kinase-3 (GSK-3) as a microtubule-associated protein tau kinase that can incorporate 4 mol of phosphates into 1 mol of tau protein and cause its electrophoretic mobility shift in sodium dodecyl sulfate gels, a unique property characteristic of paired helical filament-associated pathological tau (PHF-tau) in Alzheimer's disease brains. In this report, we identified TPPKS(p)PSAAK and SPVVSGDTS(p)PR as two phosphorylation site sequences phosphorylated by kinase FA/GSK-3 in tau using peptide sequence analysis and sequential manual Edman degradation for radiosequencing. When mapping with human brain tau sequence, we further identified Ser235-Pro and Ser404-Pro as the two major phosphorylation sites according to the numbering of the longest tau isoform. Ser235 and Ser404 have been reported as two of the major abnormal phosphorylation sites in PHF-tau. Taken together, the results provide initial evidence that protein kinase FA/GSK-3 may represent one of the Ser-Pro motif-directed tau kinases involved in the abnormal phosphorylation of pathological PHF-tau in Alzheimer's disease brain.

Alzheimer Disease↗

PubMed1993

Identification and characterization of protein kinase FA/glycogen synthase kinase 3 in clathrin-coated brain vesicles.

Mg-ATP-dependent protein phosphatase activating factor [kinase FA/glycogen synthase kinase 3 (GSK-3)] has been identified in highly purified clathrin-coated vesicles (CCVs) isolated from pig brain. Kinase FA was found to exist in an inactive state but can be activated by 1% Triton X-100 or 1 M Tris-HCl extraction in brain CCVs. Activation of kinase FA in CCVs is due to disassociation of the kinase from CCVs as demonstrated on sucrose density-gradient ultracentrifugation and Sepharose CL-4B gel filtration. Using purified brain CCVs as substrates, kinase FA enhanced the endogenous phosphorylation of assembly protein complexes in the molecular weight range of 100,000-130,000 severalfold, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. Comparisons with well-defined brain CCV-associated endogenous protein kinases such as pp50 kinase/AP50 and casein kinase 2 provide evidence that kinase FA/GSK-3 represents a third potent and unique CCV-associated protein kinase distinctly different from the previously described CCV protein kinases, suggesting the possible involvement of kinase FA in the regulation of CCV functions in the brain. The results also support the notion that protein kinase FA is involved in cell surface signal transduction in the CNS.

Animals↗

PubMed1992

Cyclic inhibition-potentiation of the crosslinking of synapsin I with brain microtubules by protein kinase FA (an activator of ATP.Mg-dependent protein phosphatase).

The ATP.Mg-dependent type-1 protein phosphatase activating factor (FA) was identified as a protein kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton and is believed to be involved in the modulation of neurotransmission. More importantly, more than 90% of the phosphates in 32P-synapsin I phosphorylated by FA could be removed by the activated ATP.Mg-dependent type-1 protein phosphatase and the synapsin I phosphatase activity was found to be strictly FA-dependent. Functional study further revealed that as a synapsin I kinase, factor FA could phosphorylate synapsin I and thereby inhibits crosslinking of synapsin I with tubulin, while as a synapsin I phosphatase activator, FA could promote the crosslinking copolymerization of synapsin I with tubulin. Taken together, the results provide initial evidence that a cyclic modulation of the crosslinking copolymerization of synapsin I with brain microtubules can be controlled by factor FA, representing an efficient cyclic cascade control mechanism for the regulation of axonal transport process during neurotransmission.

Animals↗

PubMed1992

The mechanism of activation of protein kinase FA (the activator of type-1 protein phosphatase) in brain synaptosomes.

The ATP.Mg-dependent type-1 protein phosphatase and its activating factor (protein kinase FA) were identified to exist in brain synaptosome. The inactive protein phosphatase was found to exist in the synaptosomal cytosol whereas its activating factor (protein kinase FA) was present in the synaptosomal membrane, indicating that the inactive protein phosphatase and its activating factor FA are localized in two separate subcellular compartments. The membrane-bound FA was found to exist in two forms; approximately 75% of FA is inactive and trypsin-resistant, whereas 25% of FA is active and trypsin-labile. When membranes were incubated with exogenous phospholipase C, the inactive/trypsin-resistant FA could be activated and sequestered to become the active/trypsin-labile FA in a time- and dose-dependent manner. Taken together, the results provide initial evidence that the activation-sequestration of membrane-bound protein kinase FA may represent one mode of control modulating the activity of protein kinase FA and thereby to activate protein phosphatase in brain synaptosome, representing an efficient regulatory mechanism for regulating neurotransmission in the central nervous system.

Adenosine Triphosphate↗

PubMed1992

Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells.

Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell.

Animals↗