Phosphorylation sites of protein kinase C delta in H2O2-treated cells and its activation by tyrosine kinase in vitro.

Protein kinase C delta (PKC delta) is normally activated by diacylglycerol produced from receptor-mediated hydrolysis of inositol phospholipids. On stimulation of cells with H(2)O(2), the enzyme is tyrosine phosphorylated, with a concomitant increase in enzymatic activity. This activation does not appear to accompany its translocation to membranes. In the present study, the tyrosine phosphorylation sites of PKC delta in the H(2)O(2)-treated cells were identified as Tyr-311, Tyr-332, and Tyr-512 by mass spectrometric analysis with the use of the precursor-scan method and by immunoblot analysis with the use of phosphorylation site-specific antibodies. Tyr-311 was the predominant modification site among them. In an in vitro study, phosphorylation at this site by Lck, a non-receptor-type tyrosine kinase, enhanced the basal enzymatic activity and elevated its maximal velocity in the presence of diacylglycerol. The mutation of Tyr-311 to phenylalanine prevented the increase in this maximal activity, but replacement of the other two tyrosine residues did not block such an effect. The results indicate that phosphorylation at Tyr-311 between the regulatory and catalytic domains is a critical step for generation of the active PKC delta in response to H(2)O(2).

Domain structure of tropomodulin: distinct properties of the N-terminal and C-terminal halves.

The structure of tropomodulin, the unique capping protein for the pointed end (the slow-growing end) of an actin filament, was studied. An improved Escherichia coli expression system for chicken E-tropomodulin was established and tropomodulin was prepared, Tmod (N39), in which 15 amino acid residues from the original C-terminus are deleted at the DNA level. This expression and purification system accidentally co-produces an 11-kDa fragment with the original N-terminus (N11). By applying limited proteolysis to Tmod (N39), a 20-kDa C-terminal fragment (C20) was obtained. The limited proteolysis data, as well as the fluorescence spectrometry and CD analyses of Tmod (N39), C20 and N11, revealed that tropomodulin is an alpha-helical protein that consists of two distinct domains. The C-terminal half (20 kDa) is resistant to proteolysis, which suggests that this domain is tightly folded. In contrast, the N-terminal half is susceptible to proteolysis, indicating that in solution this half is likely to be extended or to form a highly flexible structure. Cross-linking experiments with glutaraldehyde indicated that Tmod (N39) and N11 can form complexes with tropomyosin, whereas C20 cannot. This confirms the previous report that the site(s) of interaction with tropomyosin resides in the N-terminal 11-kDa region of tropomodulin.

The pharmacokinetics of trinitroglycerin and its metabolites in patients with chronic stable angina.

AIMS: To study the pharmacokinetics of trinitroglycerin (GTN) and its four metabolites in angina patients during their transient use of transdermal GTN tape following intravenous administration of GTN. METHODS: Four patients received a GTN tape following intravenous administration of 0.1 microg kg-1 min-1 GTN, and the other four patients received two GTN tapes following intravenous administration of 0.2 microg kg-1 min-1 GTN. RESULTS: Plasma concentrations of GTN in both groups during tape application showed a slight decrease for the hour after the application of the tape and then were con- stant for 24 h. In contrast, the concentrations of dinitroglycerins (GDNs) and mononitroglycerins (GMNs) depended on the duration of previous intravenous administration of GTN. Neither significant cardiovascular changes nor undesirable complications were observed during the study. CONCLUSIONS: The results suggest that appropriate replacement of intravenous GTN administration with transdermal tape application could maintain a therapeutic GTN level.

Identification of high-risk breast cancer patients from genetic changes of their tumors.

To identify the genetic prognostic markers for breast cancer, we analyzed loss of heterozygosity (LOH) at 11p, 16q, 17p, 17q, and 18q, as well as amplification of the ERBB2, INT2, and MYC genes, in 131 patients with breast carcinoma, 49 of whom had lymph node involvement, but none of whom had distant metastases. Among the several chromosome arms tested, LOH at 17q was correlated with lymph node metastasis. Amplification of the ERBB2, MYC, and INT2 genes was found more frequently in tumors from patients with lymph node metastases than in tumors from those without lymph node metastases. Univariate analysis demonstrated that LOH at 17q and INT2 amplification were factors influencing disease-free survival (DFS). A multivariate analysis was performed on 89 tumors that were able to be evaluated for both LOH at 17q and INT2 amplification, and the results showed that patients who had tumors with these genetic changes were more likely to have a poor prognosis. The findings of this study suggest that investigating genetic changes, in addition to conventional clinicopathologic factors, may contribute to defining groups of breast cancer patients with differences in prognosis.

Phosphorylation of the myristoylated protein kinase C substrate MARCKS by the cyclin E-cyclin-dependent kinase 2 complex in vitro.

The myristoylated alanine-rich C-kinase substrate (MARCKS) purified from brain was recently characterized as a proline-directed kinase(s) substrate in vivo [Taniguchi, Manenti, Suzuki and Titani (1994) J. Biol. Chem. 269, 18299-18302]. Here we have investigated the phosphorylation of MARCKS by various cyclin-dependent kinases (Cdks) in vitro. We established that Cdk2, Cdk4 and, to a smaller extent, Cdk1 that have been immunoprecipitated from cellular extracts phosphorylate MARCKS. Comparison of MARCKS phosphorylation by protein kinase C (PKC) and by the purified cyclin E-Cdk2 complex suggested that two residues were phosphorylated by Cdk2 under these conditions. To identify these sites, Cdk2-phosphorylated MARCKS was digested with lysyl endoprotease and analysed by electrospray MS. Comparison with the digests obtained from the unphosphorylated protein demonstrated that two peptides, Gly12-Lys30 and Ala138-Lys152, were phosphorylated by cyclin E-Cdk2. The identity of these peptides was confirmed by automatic Edman degradation. On the basis of the consensus phosphorylation sequence described for Cdk2, and on MS/MS analysis of the Ala138-Lys152 peptide, we concluded that Ser27, one of the phosphorylation sites identified in vivo, and Thr150 were the Cdk2 targets in vitro. None of the other sites described in vivo were phosphorylated in these conditions. Interestingly, a preliminary phosphorylation of MARCKS by PKC improved the initial rate of phosphorylation by Cdk2 without modifying the number of sites concerned. In contrast, phosphorylation of MARCKS by Cdk2 did not significantly affect further phosphorylation by PKC.

Identification of the calmodulin-binding domain of neuron-specific protein kinase C substrate protein CAP-22/NAP-22. Direct involvement of protein myristoylation in calmodulin-target protein interaction.

Various proteins in the signal transduction pathways as well as those of viral origin have been shown to be myristoylated. Although the modification is often essential for the proper functioning of the modified protein, the mechanism by which the modification exerts its effects is still largely unknown. Brain-specific protein kinase C substrate, CAP-23/NAP-22, which is involved in the synaptogenesis and neuronal plasticity, binds calmodulin, but the protein lacks any canonical calmodulin-binding domain. In the present report, we show that CAP-23/NAP-22 isolated from rat brain is myristoylated and that the modification is directly involved in its interaction with calmodulin. Myristoylated and non-myristoylated recombinant proteins were produced in Escherichia coli, and their calmodulin-binding properties were examined. Only the former bound to calmodulin. Synthetic peptides based on the N-terminal sequence showed similar binding properties to calmodulin, only when they were myristoylated. The calmodulin-binding site narrowed down to the myristoyl moiety together with a nine-amino acid N-terminal basic domain. Phosphorylation of a single serine residue in the N-terminal domain (Ser5) by protein kinase C abolished the binding. Furthermore, phosphorylation of CAP-23/NAP-22 by protein kinase C was also found myristoylation-dependent, suggesting the importance of myristoylation in protein-protein interactions.

Presence of conserved domains in the C-terminus of MARCKS, a major in vivo substrate of protein kinase C: application of ion trap mass spectrometry to the elucidation of protein structures.

MARCKS, the major protein kinase C substrate in various cells and tissues, binds to calmodulin, acidic membrane phospholipids, and actin filaments, and these interactions are regulated by protein phosphorylation. We have previously analyzed MARCKS purified from bovine brain using capillary liquid chromatography/electrospray mass spectrometry and found that the protein structure differed significantly from that deduced from cDNA sequences [Taniguchi, H., Manenti, S., Suzuki, M., and Titani, K. (1994) J. Biol. Chem. 269, 18299-18302]. Moreover, the alignment of the protein from various species showed a lack of any conserved sequences in the C-terminal half of the molecule. This prompted us to reexamine the C-terminal amino-acid sequence of bovine MARCKS. The purified protein was digested with lysyl endoprotease, and the obtained C-terminal peptide was further digested with either Staphylococcus V8 protease or NTCB. The small peptides thus obtained were analyzed by liquid chromatography/electrospray/tandem mass spectrometry. This combined with gas-phase Edman sequencing allowed us to determine the C-terminal primary structure. The sequence obtained differed significantly from that reported previously, and the comparison with other species revealed the presence of a novel conserved domain in the C-terminal region of MARCKS.

The C-terminal conserved domain of MARCKS is phosphorylated in vivo by proline-directed protein kinase. Application of ion trap mass spectrometry to the determination of protein phosphorylation sites.

MARCKS, the major protein kinase C substrate in various cells and tissues, binds to calmodulin, acidic membrane phospholipids, and actin filaments, and these interactions are regulated by protein phosphorylation. We have previously shown that MARCKS purified from bovine brain is phosphorylated not only by protein kinase C but also by so-called proline-directed protein kinases in the well conserved N-terminal half of the molecule (Taniguchi, H., Manenti, S., Suzuki, M., and Titani, K. (1994) J. Biol. Chem. 269, 18299-18302). Although the presence of other phosphorylation sites in the C-terminal peptide was also noticed, the ambiguity in the C-terminal domain of the bovine protein hampered a more detailed analysis. In the present study, we analyzed MARCKS purified from rat brain by electrospray ionization/ion trap mass spectrometry. The results obtained revealed two additional novel phosphorylation sites in the C-terminal region. Both phosphorylation sites (Ser291 and Ser299) are immediately followed by proline, suggesting that these sites are also phosphorylated by the proline-directed protein kinase(s). Since Ser299 is within the C-terminal domain, which is well conserved among various species, the function of the domain, whatever it is, seems to be controlled by phosphorylation.

MARCKS, a major protein kinase C substrate, assumes non-helical conformations both in solution and in complex with Ca2+-calmodulin.

MARCKS, a major cellular substrate for protein kinase C, plays important roles in various cellular functions and its functions are regulated by calmodulin. We have studied the conformational properties of recombinant human MARCKS in solution and in complex with calmodulin. Circular dichroism (CD) spectra showed a high content of random coil in physiological solution. When MARCKS or MARCKS-derived calmodulin-binding peptide was complexed with Ca2+-calmodulin, little change was observed in the CD spectra, suggesting that MARCKS binds with calmodulin in a non-helical conformation, which is unique among the calmodulin-binding proteins.

Specific binding of acidic phospholipids to microtubule-associated protein MAP1B regulates its interaction with tubulin.

Microtubule-associated protein MAP1B, a major neuronal cytoskeletal protein, is expressed highly during the early stage of brain development and is thought to play an important role in brain development. Although it has been shown that MAP1B localizes both in cytosol and particulate fractions, the underlying molecular mechanism in the membrane localization has yet to be elucidated. In the present study, we show that MAP1B purified from young rat brain can bind to acidic phospholipids, such as phosphatidylserine, but not to a neutral phospholipid, phosphatidylcholine. Furthermore, the binding of MAP1B to taxol-stabilized microtubules was inhibited by the addition of phosphatidylserine or phosphatidylinositol. The addition of phosphatidylcholine showed no effect on the binding of MAP1B to the microtubules. A 120-kDa microtubule-binding fragment of MAP1B was also released from microtubules by the addition of acidic phospholipids. Synthetic peptides derived from the C-terminal half of the tubulin-binding domain, but not that corresponding to the N-terminal half, bound to acidic phospholipids specifically. These results suggest that MAP1B binds to biological membranes through its tubulin-binding site, and the binding may play a regulatory role in MAP1B-microtubule interaction.

Effect of a high alpha-linolenate and high linoleate diet on membrane-associated enzyme activities in rat brain--modulation of Na+, K+- ATPase activity at suboptimal concentrations of ATP.

Semi-purified diets supplemented with either a high alpha-linolenate (n - 3) (perilla) oil or a high linoleate (n - 6) (safflower) oil were fed to rats through two generations. Rats fed safflower oil showed a decrease in docosahexaenoic acid (n - 3) and a compensatory increase in docosapentaenoic acid (n - 6) in all the brain regions and organelles examined, when compared with rats fed perilla oil. As reported previously, the safflower oil-fed rats exhibited inferior learning ability compared with the perilla oil-fed rats (N. Yamamoto et al., J. Lipid Res. 28, 144 (1987)). Using brains of rats in these dietary groups, the activities of several enzymes, Na+ , K+-ATPase, Ca2+-ATPase, 5'-nucleotidase, 2',3'-cyclic nucleotide phosphodiesterase, acetylcholinesterase, and choline acetyltransferase in membranes, were compared. The 5'-nucleotidase activity in cortex and hippocampus, and the Na+, K+-ATPase activity in myelin decreased slightly but significantly in the safflower oil group. None of the other membrane-associated enzyme activities in all the brain regions and organelles examined was affected significantly by the dietary fatty acids under optimal assay conditions in vitro. However, in the safflower oil group, the Na+, K+-ATPase activity of synaptosomes at a suboptimal concentration of ATP was 78% that in the perilla oil group. These results suggest that relatively large changes in the proportions of n - 3 and n - 6 polyunsaturated fatty acids in brain membranes caused by dietary manipulation do not provoke significant alterations in most membrane-bound enzyme activities. However, a small but significant change in Na+, K+-ATPase activity at a suboptimal concentration of ATP may be implicated in the altered learning behavior reported earlier.

CAF versus CAF plus Medroxyprogesterone Acetate for Treatment of Liver Metastases of Breast Cancer.

A controlled randomized trial was conducted to compare the effectiveness of a CAF therapy including cyclophosphamide (CPA), adriamycin (ADR) and 5-fluorouracil (5FU) with that of CAF plus medroxyprogesterone acetate (MPA) therapy including CPA, ADR and 5FU plus MPA for the treatment of liver metastases from breast cancer. A total of 34 patients with unresectable liver metastases from breast cancer were divided into two treatment groups(CAF and CAF + MPA)with stratification for estrogen receptor status. The response rate was 13%(2PR in 16 patients)for CAF therapy and 22% (1 CR and 3 PR in 18 patients) for CAF plus MPA therapy. There was no significant difference in response rates, median survival periods and survival rates between the two treatment groups. However, CAF therapy had significantly more toxicity than did CAF plus MPA therapy. The findings of this study suggested that CAF plus MPA therapy is a well-tolerated and effective treatment for patients with liver metastases of breast cancer.

Conventional Dose CAF Therapy versus Low Dose Adriamycin Therapy in the Treatment of Advanced Breast Cancer.

A controlled randomized trial was conducted to compare the effectiveness of a conventional dose of CAF therapy with that of a low dose of adriamycin (ADR) therapy for the treatment of advanced Breast Cancer. The doses of medication for the conventional CAF therapy were 100 mg/body of cyclophosphamide (CPA) p.o. daily fort wo weeks, 30 mg/m(2) of ADR and 500 mg/m(2) of 5-fluorouracil (5-FU) i.v. on days 1 and 8 for induction, and 200 mg/body of 5-FU and 20 mg/body of tamoxifen (TAM) p.o. daily for maintenance. Those for the low dose ADR therapy were 15mg/m(2) of ADR i.v. at two-week intervals for one year and 200 mg/body of 5-FU and 20 mg/body of TAM p.o. daily. Eighty patients were entered in this trial. All patients were randomly divided into two groups with stratification for estrogen receptor status. Of 78 patients among them, 38 undergoing the CAF therapy and 40 undergoing the low dose ADR therapy, were evaluated for efficacy assesment. The background factors analyzed were well balanced in both groups. The response rate was 47%(6CR, 12PR out of 38) in the CAF group and 43% (3CR, 14PR out of 40) in the low dose ADR group. There was no significant difference in response rates and survival rates as determined by the Kaplan Meier method between the two groups. The CAF therapy had significantly more toxicity than the low dose ADR therapy. Therefore, it was concluded that this low dose ADR therapy can be regarded as a treatment of choice for advanced breast cancer.

Lysophosphoinositide-specific phospholipase C in rat brain synaptic plasma membranes.

A membrane preparation from rat brain catalyzed the hydrolysis of [2-3H]glycerol-labeled lysophosphatidylinositol (lysoPI) to yield monoacylglycerol (MG) and inositolphosphates. This phospholipase C activity had an optimal pH of 8.2. The membrane preparation did not require the addition of Ca2+ for its maximum activity, but the activity was inhibited by addition of 0.1 mM EDTA to the assay mixture and was restored by simultaneous addition of 0.2 mM Ca2+. The activity was found to be localized in synaptic plasma membranes prepared by Ficoll and Percoll density gradients. The phospholipase C was highly specific for lysoPI; diacylglycerol formation from phosphatidylinositol, and MG formation from lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylserine were below 5% of that observed with lysoPI under the conditions used. We concluded that there is a pathway for phosphatidylinositol metabolism in brain synaptic membranes which is different from the well-characterized phosphoinositide-specific phospholipase C pathway.

Ossification of the ligamentum teres as a possible mechanism of lateral subluxation in coxopathy associated with ankylosing spinal hyperostosis.

The cases are reported of two patients with coxopathy associated with ankylosing spinal hyperostosis who showed ossification of the ligamentum teres. It is suggested that considerable ossification at the medial acetabular floor including the ligamentum teres can explain the mechanism of development of coxopathy with lateral subluxation of the femoral head which is often seen in patients with ankylosing spinal hyperostosis.

Heterophile Hanganutziu-Deicher antibodies in sera of patients with Kawasaki diseases.

The heterophile antibody levels in sera from patients with Kawasaki disease (49 sera from 39 cases) were measured by sheep erythrocytes (SRBC) agglutination and radioimmunoassay in microplates coated with Hanganutziu-Deicher (H-D) antigen-active glycosphingolipid, equine hematoside. The antibody levels were low in the first week of illness, increased rapidly in the 2nd week, and thereafter gradually decreased. The SRBC agglutination titers and H-D antibody titers of sera from patients with Kawasaki disease from week 2 to 8 of illness were significantly higher than those of healthy children (44 sera) and normal cord blood (13 sera).

Application of a gas chromatography mass spectrometry computer system for clinical diagnosis.

Currently, one of the major clinical problems is diagnosis of inherited metabolic disorders in neonates which are caused by a defect of specific enzymes. In order to diagnose such diseases at an earlier stage, we have developed a new mass screening method, consisting of a gas chromatography mass spectrometry computer system with a newly developed 'Diagnosis Computer Program'. In this program, the chromatographic pattern of the urine of a patient with an inherited metabolic disorder is compared with that of a normal person. Internal standards are applied to simplify the comparison. The total ion current chromatogram is drawn with the relative retention times plotted on the horizontal axis and the relative intensities on the vertical axis. Diseases are diagnosed by characteristics and quantity of abnormal compounds in the urine. Reference to the stored mass spectra of abnormal compounds by a library search enables the diagnosis to be more precise. Using this program, twenty-six inherited metabolic disorders are routinely diagnosed. The diagnosis of tyrosinosis is used here as an example.