PubMed Health⌕ Search

Biomedical subjects

S D Yang

Publications and source records attributed to S D Yang.

At least 37 records · Page 2Linked to original sources
PubMed1997

Heat stress induces tyrosine phosphorylation/activation of kinase FA/GSK-3 alpha (a human carcinoma dedifferentiation modulator) in A431 cells.

Exposure of A431 cells to a rapid temperature increase from 37 degrees to 46 degrees C could induce an increased expression (approximately 200% of control) and tyrosine phosphorylation/activation (approximately 300% of control) of protein kinase FA/ glycogen synthase kinase-3 alpha (kinase FA/GSK-3 alpha) in a time-dependent manner, as demonstrated by an anti-kinase FA/GSK-3 alpha immunoprecipitate kinase assay and by immunoblotting analysis with anti-kinase FA/GSK-3 alpha and anti-phosphotyrosine antibodies. The heat induction on the increased expression of kinase FA/GSK-3 alpha could be blocked by actinomycin D but not by genistein. In contrast, the heat induction on tyrosine phosphorylation/activation of kinase FA/GSK-3 alpha could be blocked by genistein or protein tyrosine phosphatase, indicating that heat stress induces a dual control mechanism, namely, protein expression and subsequent tyrosine phosphorylation to cause cellular activation of kinase FA/GSK-3 alpha. Taken together, the results provide initial evidence that kinase FA/GSK-3 alpha represents a newly described heat stress-inducible protein subjected to tyrosine phosphorylation/activation, representing a new mode of signal transduction for the regulation of this human carcinoma dedifferentiation modulator and a new mode of heat induction on cascade activation of a protein kinase.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

Reversible tyrosine phosphorylation/dephosphorylation of proline-directed protein kinase FA/glycogen synthase kinase-3alpha in A431 cells.

Modulation of protein kinase FA/glycogen synthase kinase-3alpha (kinase FA/GSK-3alpha) by reversible tyrosine phosphorylation/dephosphorylation was investigated. In addition to genistein, other protein tyrosine kinase (PTK) inhibitors, such as tyrphostin A47 and B42, also could induce tyrosine dephosphorylation and inactivation of kinase FA/GSK-3alpha in A431 cells, and this process was found to be reversible. Pretreatment of the cells with 100 microM orthovanadate, a protein tyrosine phosphatase (PTP) inhibitor, could diminish significantly the effects of PTK inhibitors on both enzyme activity and phosphotyrosine content of the kinase, suggesting that the PTK inhibitors induced tyrosine dephosphorylation/inactivation of this kinase is mediated by orthovanadate-sensitive PTP(s) in A431 cells. Moreover, the phosphotyrosine moiety of kinase FA/GSK-3alpha was found to be highly turned over in resting cells. Interestingly, we found that the less active, tyrosine-dephosphorylated form of kinase FA/GSK-3alpha immunoprecipitated from genistein-treated cells was able to reactivate partially with concomitant rephosphorylation of tyrosine residue in vitro. Taken together, these findings demonstrate that tyrosine phosphorylation and concomitant activation of kinase FA/GSK-3alpha can be carried out both in vitro and in vivo and an in vivo phosphatase activity may function in antagonism to PTK activation of kinase FA/GSK-3alpha.

Animals↗
PubMed1997

Mono(ADP-ribosyl)transferase genes and diabetes in NOD mice. Is there a relationship?

The answer to the question posed by the title (is there a relationship between aberrant Art gene expression and IDDM pathogenesis in NOD mice?) remains elusive. Conclusions are currently based almost entirely upon analysis of mRNA transcript levels rather than on T-cell-specific mono-ADP ribosylation activities. Our unpublished data, as well as data published in abstract form by Dr. L. Chatenoud and colleagues (48) indicate that gene transcription is not impaired in splenic leukocytes of older NOD mice, including those with spontaneous IDDM development. Based upon the limited data showing that there may be reduced expression of Art gene products in the earliest T cell immigrants from the NOD thymus, one would have to surmise that If there is a regulatory defect, it may be in allowing single positive thymic T cells to emigrate before they are fully mature. Therefore, development of anti-Art monoclonal antibody together with further studies regarding functions of mono(ADP-ribosyl)transferase in immunoregulation of different subpopulation of T-cells, may finally resolve the role that altered mono(ADP-ribosyl)transferase activities play in the pathogenesis of IDDM in NOD mice.

ADP Ribose Transferases↗
PubMed1996

Calphostin C induces tyrosine dephosphorylation/inactivation of protein kinase FA/GSK-3 alpha in a pathway independent of tumor promoter phorbol ester-mediated down-regulation of protein kinase C.

The signal transduction mechanism of protein kinase FA/GSK-3 alpha by tyrosine phosphorylation in A431 cells was investigated using calphostin C as an inhibitor for protein kinase C (PKC). Kinase FA/GSK-3 alpha could be tyrosine-dephosphorylated and inactivated to approximately 10% of control in a concentration-dependent manner by 0.1-10 microM calphostin C (IC50, approximately 1 microM), as demonstrated by immunoprecipitation of kinase FA/GSK-3 alpha from cell extracts, followed by phosphoamino acid analysis and by immunodetection in an antikinase FA/GSK-3 alpha immunoprecipitate kinase assay. In sharp contrast, down-regulation of PKC by 0.05 microM calphostin C (IC50, approximately 0.05 microM for inhibiting PKC in cells) or by tumor promoter phorbol ester TPA was found to have stimulatory effect on the cellular activity of kinase FA/GSK-3 alpha, when processed under identical conditions. Furthermore, TPA-mediated down-regulation of PKC was found to have no effect on calphostin C-mediated tyrosine dephosphorylation/inactivation of kinase FA/GSK-3 alpha. Taken together, the results provide initial evidence that the PKC inhibitor calphostin C may induce tyrosine dephosphorylation/inactivation of kinase FA/GSK-3 alpha in a pathway independent of TPA-mediated down-regulation of PKC, representing a new mode of signal transduction for the regulation of this multisubstrate/multifunctional protein kinase by calphostin C in cells. Since kinase FA/GSK-3 alpha is a possible carcinoma dedifferentiation/progression-promoting factor, the results further suggest calphostin C as a potential anticancer drug involved in blocking carcinoma dedifferentiation/progression, possibly via inactivation of protein kinase FA/GSK-3 alpha in tumor cells.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

Okadaic acid, sphingosine, and phorbol ester reversibly modulate heat induction on protein kinase FA/GSK-3 alpha in A431 cells.

Exposure of A431 cells to a rapid and sudden increase from 37 degrees C to 46 degrees C for 30 min could induce an increase in protein level and cellular activity of protein (kinase FA/GSK-3 alpha) up to approximately 200% of control level. However, when cells were first treated with 500 nM tumor promoter phorbol ester TPA at 37 degrees C for 30 min to activate cellular protein kinase C (PKC) or with 400 nM okadaic acid at 37 degrees C for 30 min to inhibit cellular protein phosphatases followed by heat shock at 46 degrees C for another 30 min, the heat induction on kinase FA/GSK-3 alpha was found to be completed blocked. In sharp contrast, when cells were first treated with 1 microM TPA at 37 degrees C for 24 h or with 5 microM sphingosine at 37 degrees C for 30 min to down-regulate cellular PKC, the heat induction on kinase FA/GSK-3 alpha was found to be reversely promoted up to approximately 250% of control level, demonstrating that kinase FA/GSK-3 alpha may not represent a constitutively active/mitogen-inactivated protein kinase as previously conceived. Taken together, the results provide initial evidence that TPA/sphingosine and okadaic acid could reversibly modulate the heat induction on kinase FA/GSK-3 alpha in A431 cells, suggesting that phosphorylation/dephosphorylation mechanisms are involved in the regulation of the heat-shock induction of kinase FA/GSK-3 alpha, representing a new mode of signal transduction for the regulation of this multisubstrate protein kinase and a new mode of signaling pathway modulating the heat-induction process.

Amino Acid Sequence↗
PubMed1996

Overexpression of protein kinase FA/GSK-3 alpha (a proline-directed protein kinase) correlates with human hepatoma dedifferentiation/progression.

Computer analysis of protein phosphorylation sites sequence revealed that transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of the proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3 alpha (kinase F(A)/GSK-3 alpha) (a member of PDPK family) has been optimized for human hepatoma and used to demonstrate for the first time significantly increased (P < 0.01) activity in poorly differentiated SK-Hep-1 hepatoma (24.2 +/- 2.8 units/mg) and moderately differentiated Mahlavu hepatoma (14.5 +/- 2.2 units/mg) when compared to well differentiated Hep 3B hepatoma (8.0 +/- 2.4 units/mg). Immunoblotting analysis revealed that increased activity of kinase FA/GSK-3 alpha is due to overexpression of the protein. Elevated kinase FA/GSK-3 alpha expression in human hepatoma biopsies relative to normal liver tissue was found to be even more profound. This kinase appeared to be fivefold overexpressed in well differentiated hepatoma and 13-fold overexpressed in poorly differentiated hepatoma when compared to normal liver tissue. Taken together, the results provide initial evidence that overexpression of kinase FA/GSK-3 alpha is involved in human hepatoma dedifferentiation/progression. Since kinase FA/GSK-3 alpha is a PDPK, the results further support a potential role of this kinase in human liver tumorigenesis, especially in its dedifferentiation/progression.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

Activation of the ATP.Mg-dependent type 1 protein phosphatase by the Fe2+/ascorbate system.

The ATP.Mg-dependent type 1 protein phosphatase is inactive as isolated but can be activated in several different ways. In this report, we show that the phosphatase can also be activated by the Fe2+/ascorbate system. Activation of the phosphatase requires both Fe2+ ion and ascorbate and the level of activation is dependent on the concentrations of Fe2+ ion and ascorbate. In the presence of 20 mM ascorbate, the Fe2+ ion concentrations required for half-maximal and maximal activation are about 0.3 and 3 mM, respectively. Several common divalent metal ions, including CO2+, Ni2+, Cu2+, Mg2+, and Ca2+ ions, cannot cooperate with ascorbate to activate the phosphatase, and SH-containing reducing agents such as 2-mercaptoethanol and dithiothreitol cannot cooperate with Fe2+ ion to activate the phosphatase, indicating that activation of the phosphatase by the Fe2+/ascorbate system is a specific process. Moreover, H2O2, a strong oxidizer, could significantly diminish the phosphatase activation by the Fe2+/ascorbate system, suggesting that reduction mechanism other than SH-SS interchange is a prerequisite for the Fe2+/ascorbate-mediated phosphatase activation. Taken together, the present study provides initial evidence for a new mode of type 1 protein phosphatase activation mechanism.

Animals↗
PubMed1996

Hyperglycemia induced activation of type-1 protein phosphatase activator (kinase FA) in perfused human placenta.

We report the identification of type-1 protein phosphatase activating factor (kinase FA), a unique biologic mediator for both insulin and epidermal growth factors in the human placenta. The activity of kinase F, was found to be extremely labile in the unperfused placenta. Fresh term placentas lost more than 50% of the total kinase FA activity within 6 hours when exposed to air of incubated in medium but not perfused. In contrast, the activity of kinase FA was stable when the human term placenta was dually perfused. This indicates that placental dual perfusion is a useful method for studying protein phosphorylation-dephosphorylation involved in signal transduction. When fresh placentas were perfused with media containing glucose at 141 +/- 10, 242 +/- 12 and 436 +/- 20 mg/dL, kinase FA activity was stimulated several-fold in a glucose concentration-dependent manner when compared with control levels at delivery. The results suggest that hyperglycemia-mediated activation may represent a previously unknown control mechanisms for the regulation of protein kinase FA. The results also suggest that human placental perfusion is a good in vitro system for studying signal transduction mechanisms involved in hormonal actions and metabolic regulation.

Analysis of Variance↗
PubMed1995

Phosphorylation/activation of phosphorylase b kinase by cAMP/Ca2(+)-independent, autophosphorylation-dependent protein kinase.

Phosphorylase b kinase from rabbit skeletal muscle can be phosphorylated and activated by a cyclic nucleotide- and Ca2(+)-independent protein kinase previously identified as an autophosphorylation-dependent multifunctional protein kinase (auto-kinase) from brain and liver (Yang et al., J. Biol. Chem. 262, 7034-7040 (1987) and Yang et al. J. Biol. Chem. 262, 9421-9427 (1987)). This independent kinase phosphorylates both alpha and beta subunits of phosphorylase b kinase and results in a approximately 5-fold activation of the kinase when 0.55 and 0.5 mol of phosphate are incorporated into the alpha and beta subunits, respectively. Activation of phosphorylase b kinase catalyzed by auto-kinase is about 70% of that observed with cAMP-dependent protein kinase. Analysis of phosphopeptide maps of alpha and beta subunits further reveals that both kinases phosphorylate almost the same sites on both alpha and beta subunits, suggesting that activation of phosphorylase b kinase by the two kinases may be through a common molecular action mechanism. Taken together with the previous result that auto-kinase can inactivate glycogen synthase, the present study provides initial evidence that a coordinate control mechanism for simultaneous regulation of glycogenolysis and glycogenesis can be modulated by autophosphorylation-dependent protein kinase in a cAMP- and Ca2(+)-independent pathway, representing a new mode of control mechanism for the regulation of glycogen metabolism in cells.

Animals↗
PubMed1995

Overexpression of cellular activity and protein level of protein kinase FA/GSK-3 alpha correlates with human thyroid tumor cell dedifferentiation.

Computer analysis of protein phosphorylation sites sequence revealed that transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of the proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3 alpha (kinase FA/GSK-3 alpha) (a member of the PDPK family) has been optimized for human thyroid tissue and used to demonstrate for the first time significantly increased (P < 0.001) activity in thyroid carcinoma (24.2 +/- 2.8 units/mg of protein) (n = 7), thyroid adenoma (14.5 +/- 2.2 units/mg of protein) (n = 6), and thyroid hyperplasia (8.0 +/- 2.4 units/mg of protein) (n = 5) when compared to five normal controls (4.1 +/- 1.8 units/mg of protein). Immunoblotting analysis further revealed that increased activity of kinase FA/GSK-3 alpha in thyroid tumor cells is due to overexpression of the protein synthesis of the enzyme. Taken together, the results provide initial evidence that overexpression of protein level and cellular activity of kinase FA/GSK-3 alpha is involved in human thyroid tumor cell dedifferentiation, supporting an association of PDPK with neoplastic transformation and tumorigenesis. Since kinase FA/GSK-3 alpha may function as a possible regulator of transcription factors/protooncogenes, kinase FA/GSK-3 alpha may therefore play an important role in thyroid cell carcinogenesis, especially in its differentiation.

Adenoma↗
PubMed1995

Dysfunction of protein kinase FA/GSK-3 alpha in lymphocytes of patients with schizophrenic disorder.

As compared to normal people, the lymphocytes of patients with schizophrenia were found to have an impairment of ATP.Mg-dependent protein phosphatase activation. More importantly, the impaired protein phosphatase activation in the lymphocytes of schizophrenic patients could be consistently and completely restored to normal by exogenous pure protein kinase FA/glycogen synthase kinase-3 alpha (kinase FA/GSK-3 alpha) (the activating factor of ATP.Mg-dependent protein phosphatase), indicating that the molecular mechanism for the impaired protein phosphatase activation in schizophrenic patients may be due to a functional loss of kinase FA/GSK-3 alpha. Immunoblotting and kinase activity analysis in an anti-kinase FA/GSK-3 alpha immunoprecipitate further demonstrate that both cellular activities and protein levels of kinase FA/GSK-3 alpha in the lymphocytes of schizophrenic patients were greatly impared as compared to normal controls. Statistical analysis revealed that the lymphocytes isolated from 37 normal people contain kinase FA/GSK-3 alpha activity in the high levels of 14.8 +/- 2.4 units/mg of cell protein, whereas the lymphocytes of 48 patients with schizophrenic disorder contain kinase FA/GSK-3 alpha activity in the low levels of 2.8 +/- 1.6 units/mg, indicating that the different levels of kinase FA/GSK-3 alpha activity between schizophrenic patients and normal people are statistically significant. Taken together, the results provide initial evidence that patients with schizophrenic disorder may have a common impairment in the protein levels and cellular activities of kinase FA/GSK-3 alpha, a multisubstrate protein kinase and a multisubstrate protein phosphatase activator in their lymphocytes.

Amino Acid Sequence↗
PubMed1995

Association of protein kinase FA/GSK-3alpha (a proline-directed kinase and a regulator of protooncogenes) with human cervical carcinoma dedifferentiation/progression.

Computer analysis of protein phosphorylation-sites sequence revealed that most transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3alpha (kinase FA/GSK-3alpha) (a particular member of PDPK family) has been optimized for human cervical tissue and used to demonstrate for the first time significantly increased (P < 0.001) activity in poorly differentiated cervical carcinoma (82.8 +/- 6.6 U/mg of protein), moderately differentiated carcinoma (36.2 +/- 3.4 U/mg of protein), and well-differentiated carcinoma (18.3 +/- 2.4 U/mg of protein) from 36 human cervical carcinoma samples when compared to 12 normal controls (4.9 +/- 0.6 U/mg of protein). Immunoblotting analysis further revealed that increased activity of kinase FA/GSK-3alpha in cervical carcinoma is due to overexpression of protein synthesis of the kinase. Taken together, the results provide initial evidence that overexpression of protein synthesis and cellular activity of kinase FA/GSK-3alpha may be involved in human cervical carcinoma dedifferentiation/progression, supporting an association of proline-directed protein kinase with neoplastic transformation and tumorigenesis. Since protein kinase FA/GSK-3alpha may function as a possible regulator of transcription factors/proto-oncogenes, the results further suggest that kinase FA/GSK-3alpha may play a potential role in human cervical carcinogenesis, especially in its dedifferentiation and progression.

Amino Acid Sequence↗
PubMed1995

Effect of Mg2+ concentrations on phosphorylation/activation of phosphorylase b kinase by cAMP/Ca(2+)-independent, autophosphorylation-dependent protein kinase.

In a previous report [Yu and Yang, Biochem. Biophys. Res. Commun. 207, 140-147 (1995)], phosphorylase b kinase from rabbit skeletal muscle was found to be phosphorylated and activated by a cyclic nucleotide- and Ca(2+)-independent protein kinase previously identified as an autophosphorylation-dependent multifunctional protein kinase (autokinase) from brain and liver [Yang et al., J. Biol. Chem. 262, 7034-7040, 9421-9427 (1987)]. In this report, the effect of Mg2+ ion concentration on the auto-kinase-catalyzed activation of phosphorylase b kinase is investigated. The levels of phosphorylation and activation of phosphorylase b kinase catalyzed by auto-kinase are found to be dependent on the concentration of Mg2+ ion used. Phosphorylation of phosphorylase b kinase at high Mg2+ ion (> 9 mM) is 2-3 times higher than that observed at low Mg2+ ion (1 mM) and this results in a further 2- to 3-fold activation of the enzyme activity at high Mg2+ ion. Analysis of the phosphorylation stoichiometry of alpha and beta subunits of phosphorylase b kinase at different Mg2+ ion concentrations further reveals that the phosphorylation level of the beta subunit remains almost unchanged, whereas the phosphorylation level of the alpha subunit increases dramatically and correlates with the increased enzyme activity. In similarity with the beta subunit, phosphorylations of myelin basic protein and histone 2A by auto-kinase are also unaffected by Mg2+ ion. Taken together, the results provide initial evidence that Mg2+ ion may specifically render the alpha subunit a better substrate for auto-kinase to cause further phosphorylation/activation of phosphorylase b kinase, representing a new mode of control mechanism for the regulation of auto-kinase involved in the phosphorylation and concurrent activation of phosphorylase b kinase.

Adenosine Triphosphate↗
PubMed1995

Tau protein kinase I/GSK-3 beta/kinase FA in heparin phosphorylates tau on Ser199, Thr231, Ser235, Ser262, Ser369, and Ser400 sites phosphorylated in Alzheimer disease brain.

Previously, tau protein kinase I/glycogen synthase kinase-3 beta/kinase FA(TPKI/GSK-3 beta/FA) was identified as a brain microtubule-associated tau kinase possibly involved in the Alzheimer disease-like phosphorylation of tau. In this report, we find that the TPKI/GSK-3 beta/FA can be stimulated to phosphorylate brain tau up to 8.5 mol of phosphates per mol of protein by heparin, a polyanion compound. Tryptic digestion of 32P-labeled tau followed by high-performance liquid chromatography and high-voltage electrophoresis/thin-layer chromatography reveals 12 phosphopeptides. Phosphoamino acid analysis together with sequential manual Edman degradation and peptide sequence analysis further reveals that TPKI/GSK-3 beta/FA after heparin potentiation phosphorylates tau on sites of Ser199, Thr231, Ser235, Ser262, Ser396, and Ser400, which are potential sites abnormally phosphorylated in Alzheimer tau and potent sites responsible for reducing microtubule binding possibly involved in neuronal degeneration. The results provide initial evidence that TPKI/GSK-3 beta/FA after heparin potentiation may represent one of the most potent systems possibly involved in the abnormal phosphorylation of PHF-tau and neuronal degeneration in Alzheimer disease brains.

Alzheimer Disease↗
PubMed1995

Differential expression of novel genes by bone marrow-derived macrophage populations.

In the present study, we have constructed a subtraction cDNA library to identify novel genes induced by IFN-gamma in GM-CSF-derived bone marrow macrophage (m phi). M theta were treated with 50 U/ml IFN-gamma for 40, 70 and 140 min to induce expression of early genes regulated by IFN-gamma, and the M phi were pooled. Poly(A)+RNA was prepared from both unactivated and IFN-gamma-stimulated m theta, and cDNA libraries were constructed in lambda ZAP. Genes expressed in common by both m theta populations were removed by subtraction using biotin-avidin precipitation of hybrid complexes. Further selection was performed by differential screening using cDNA prepared from mRNA of unactivated m phi as a probe, followed by colony hybridization to remove sister clones. Of 17 clones from which sequence information was obtained, two appeared to be identical with the murine genes, C10 (clone GM2B1) and Mac-2 (clone GM2C4) and an additional two clones had high similarity to human cDNAs encoding proteins of unknown function. cDNAs containing sequences which did not match published sequences were used to probe Northern blots prepared from both unstimulated and IFN-gamma-activated GM-CSF- and CSF-1-derived m phi. Five clones (GM1A2, GM1B4, GM1F2, GM2A12 and GM2B8) showed enhanced transcript levels following IFN-gamma treatment of GM-CSF-derived m phi, but demonstrated high constitutive transcript levels in CSF-l-derived m phi. In addition, C10 transcripts were constitutively expressed by GM-CSF-derived m phi, but not by CSF-1-derived m phi, even after activation by IFN-gamma. These data suggest that much of the functional heterogeneity of GM-CSF- and CSF-1-derived m phi resides in the differential expression of early genes specifically induced by IFN-gamma.

Animals↗
PubMed1995

F-H to AB plane angle (FABA) for assessment of anteroposterior jaw relationships.

The horizontal relationship of the jaws has been defined as the angles or distances between the reference planes of the craniofacial complex and points A and B, which are representative of the anterior limits of the denture bases. The aims of this study were (1) to examine statistically and geometrically the different cephalometric measurements which are used to indicate the A-P jaw relationship, and (2) to provide a more reliable parameter by means of comparative cephalometric analyses with various clinical examples. The APDI and Wits appraisal are parameters for evaluating the anteroposterior relationship of the dentition rather than the jaws. FH to AB plane angle (FABA) may provide not only a reliable cephalometric measurement of the anteroposterior relationship of the jaws but also a clue to the facial profile.

Adolescent↗
PubMed1995

Protein kinase FA/glycogen synthase kinase 3 alpha predominantly phosphorylates the in vivo sites of Ser502, Ser506, Ser603, and Ser666 in neurofilament.

In this report, the phosphorylation sites of neurofilament protein of medium molecular mass (NF-M) by protein kinase FA/glycogen synthase kinase 3 alpha (kinase FA/GSK-3 alpha) were determined by two-dimensional electrophoresis/TLC, phosphoamino acid analysis, HPLC, Edman degradation, and peptide sequencing. Kinase FA/GSK-3 alpha phosphorylates NF-M predominantly on serine residue. Three major tryptic phosphopeptide peaks were resolved by C18 reverse-phase HPLC. Edman degradation and peptide sequence analysis revealed that AKS(p)PVSK is the phosphorylation site sequence for the first major peak. When mapping with the amino acid sequence of neurofilament, we finally demonstrate Ser603-Pro, one of the in vivo sites in NF-M, as the major site phosphorylated by kinase FA/GSK-3 alpha. By using the same approach, we also identified the in vivo sites of Ser502-Pro, Ser506-Pro, and Ser666-Pro as the other three major sites in NF-M phosphorylated by kinase FA/GSK-3 alpha. Taken together, the results provide initial evidence that kinase FA/GSK-3 alpha may represent a physiologically relevant protein kinase involved in the vivo phosphorylation of NF-M. Because Ser502, Ser506, Ser603, and Ser666 are all flanked by a carboxyl-terminal proline residue, the results provide further evidence that FA/GSK-3 alpha may represent a proline-directed protein kinase involved in the structure-function regulation of the neuronal cytoskeletal system.

Amino Acid Sequence↗