No abstract

We cloned and characterized a novel human member of the STE20 serine/threonine protein kinase family named mst-3. Based on its domain structure, mst-3 belongs to the SPS1 subgroup of STE20-like proteins, which includes germinal center (GC) kinase, hematopoietic progenitor kinase (HPK), kinase homologous to STE20/SPS-1 (KHS), kinases responsive to stress (KRS1/ 2), the mammalian STE20-like kinases (mst1/2), and the recently published STE20/oxidant stress response kinase SOK-1. mst-3 is most closely related to SOK-1, with 88% amino acid similarity in the kinase domain. The similarity of the mst-3 kinase domain to STE20 is 42%. The mst-3 transcript is ubiquitously expressed, and the protein was found in all human, mouse, and monkey cell lines tested. An in vitro kinase assay showed that mst-3 can phosphorylate basic exogenous substrates as well as itself. Interestingly, mst-3 prefers Mn 2؉ to Mg 2؉ as a divalent cation and can use both GTP and ATP as phosphate donors. Like SOK-1, mst-3 is activated by autophosphorylation. However, a physiological stimulus of mst-3 activity was not identified. mst-3 activity does not change upon exposure to several mitogenic and stress stimuli. Overexpression of mst-3 wild-type or kinase dead protein affects neither the extracellular signal-regulated kinases (ERK1/2 or ERK6), c-Jun N-terminal kinase (JNK), p38, nor pp70S6 kinase, suggesting that mst-3 is part of a novel signaling pathway.

Focal adhesions and actin cytoskeleton are involved in cell growth, shape and movement and in tumor invasion. Mitogen-induced changes in actin cytoskeleton are accompanied by changes in the tyrosine phosphorylation of several focal adhesion proteins. In this study, we have investigated the role of RAFTK, a cytoplasmic tyrosine kinase related to focal adhesion kinase (FAK), in heregulin-mediated signal transduction in breast cancer cells. Stimulation of T47D cells with heregulin (HRG) induced the tyrosine phosphorylation of RAFTK and the formation of a multiprotein complex. Analyses of the members of the HRG-stimulated complex revealed that RAFTK is associated with p190 RhoGAP (p190), RasGAP and ErbB-2, and plays an essential role in mediating the tyrosine phosphorylation of p190 by Src. Mutation of the Src binding site within RAFTK (402) abolished the phosphorylation of p190. In addition, upon HRG stimulation of T47D cells, association of ErbB-2 with RAFTK was observed and found to be indirect and mediated by Src. Expression of wild-type RAFTK (WT) signi®cantly increased MDA-MB-435 and MCF-7 breast cancer cell invasion, while expression of the kinasemutated RAFTK-R457 (KM) or the Src binding site mutant RAFTK (402) did not a ect this cell invasion. Furthermore, HRG leads to the activation of MAP kinase which is mediated by RAFTK. These ®ndings indicate that RAFTK serves as a mediator and an integration point between the GAP proteins and HRG-mediated signaling in breast cancer cells, and implicate RAFTK involvement in the MAP kinase pathway and in breast cancer cell invasion.

No abstract

The mechanism of oxidation of 1,2-dehydro-N-acetyldopamine (dehydro NADA) was examined to resolve the controversy between our group and Andersen's group regarding the reactive species involved in beta-sclerotization. While Andersen has indicated that dehydro NADA quinone is the beta-sclerotizing agent [Andersen, 1989], we have proposed quinone methides as the reactive species for this process [Sugumaran, 1987; Sugumaran, 1988]. Since dehydro NADA quinone has not been isolated or identified till to date, we studied the enzymatic oxidation of dehydro NADA in the presence of quinone traps to characterize this intermediate. Accordingly, both N-acetylcysteine and o-phenylenediamine readily trapped the transiently formed dehydro NADA quinone as quinone adducts. Interestingly, when the enzymatic oxidation was performed in the presence of o-aminophenol or different catechols, adduct formation between the dehydro NADA side chain and the additives had occurred. The structure of the adducts is in conformity with the generation and reactions of dehydro NADA quinone methide (or its radical). This, coupled with the fact that 4-hydroxyl or amino-substituted quinones instantly transformed into p-quinonoid structure, indicates that dehydro NADA quinone is only a transient intermediate and that it is the dehydro NADA quinone methide that is the thermodynamically stable product. However, since this compound is chemically more reactive due to the presence of both quinone methide and acylimine structure on it, the two side chain carbon atoms are "activated." Based on these considerations, it is suggested that the quinone methide derived from dehydro NADA is the reactive species responsible for cross-link formation between dehydro NADA and cuticular components during beta-sclerotization.

We have recently isolated a cDNA encoding a novel human receptor-type tyrosine phosphatase, termed PTP-RO (for a protein tyrosine phosphatase receptor omicron), from 5-fluorouracil–treated murine bone marrow cells. PTP-RO is a human homologue of murine PTPλ and is related to the homotypically adhering κ and μ receptor-type tyrosine phosphatases. PTP-RO is expressed in human megakaryocytic cell lines, primary bone marrow megakaryocytes, and stem cells. PTP-RO mRNA and protein expression are upregulated upon phorbol 12-myristate 13-acetate (PMA) treatment of the megakaryocytic cell lines CMS, CMK, and Dami. To elucidate the function of PTP-RO in megakaryocytic cells and its potential involvement in the stem cell factor (SCF)/c-Kit receptor pathway, COS-7 and 293 cells were cotransfected with the cDNAs of both the c-Kit tyrosine kinase receptor and PTP-RO. PTP-RO was found to be associated with the c-Kit receptor in these transfected cells and the SCF/Kit ligand induced a rapid tyrosine phosphorylation of PTP-RO. Interestingly, these transfected cells demonstrated a decrease in their proliferative response to the SCF/Kit ligand. In addition, we assessed the association of PTP-RO with c-Kit in vivo. The results demonstrated that PTP-RO associates with c-Kit but not with the tyrosine kinase receptor FGF-R and that PTP-RO is tyrosine-phosphorylated after SCF stimulation of Mo7e and CMK cells. Antisense oligonucleotides directed against PTP-RO mRNA sequences significantly inhibited megakaryocyte progenitor proliferation. Therefore, these data show that the novel tyrosine kinase phosphatase PTP-RO is involved in megakaryocytopoiesis and that its function is mediated by the SCF/c-Kit pathway.

In accordance with o u r earlier results, quinone methide formation was confirmed t o b e t h e major pathway for the oxidation of N-acetyldopamine (NADA) by cuticle-bound enzymes from Sarcophaga bullata larvae. In addition, with t h e use of a newly developed HPLC separation condition and cuticle prepared by gentle procedures, it could b e demonstrated that 1,Z-dehydro-NADA and its dimeric oxidation products are also generated in t h e reaction mixture containing a high concentration of NADA albeit at a much lower amount than t h e NADA q u i n o n e methide water adduct, viz., N-acetylnorepinephrine (NANE). By using different buffers, it was also possible t o establish t h e accumulation of NADA quinone in reaction mixtures containing NADA and cuticle. That t h e 1,Z-dehydro-NADA formation is d u e t o t h e action of a NADA desaturase system was established by pH and temperature studies and by differential inhibition of NANE production. Of t h e various cuticle examined, adult cuticle of Locusta migratoria, presclerotized cuticle of Periplaneta americana, and white puparial cases of Drosophila rnelanogaster exhibited more NADA desaturase activity than NANE generating activity, while t h e reverse was observed with t h e larval cuticle of Tenebrio molitor and pharate pupal cuticle of Manduca sexta. These studies indicate that both NADA quinone methide and 1,Z-dehydro NADA are formed during enzymatic activation of NADA in insect cuticle. Based o n these results, a unified mechanism for @-sclerotization involving quinone methides as t h e reactive species is presented.

Abstract:Glutamate is the major excitatory neurotransmitter in the CNS. Although its role in neurons has been studied extensively, little is known about its function in astrocytes. We studied the effects of glutamate on signaling pathways in primary astrocytes. We found that the tyrosine kinase related adhesion focal tyrosine kinase (RAFTK) is tyrosine phosphorylated in response to glutamate in a time-and dose-dependent manner. This phosphorylation was pertussis toxin (PTX) sensitive and could be attenuated by the depletion of Ca 2ϩ from intracellular stores. RAFTK tyrosine phosphorylation was mediated primarily by class I/II metabotropic glutamate receptors and depends on protein kinase C (PKC) activation. Glutamate treatment of primary astrocytes also results in a significant increase in the activity of the mitogen-activated protein kinases [extracellular signal-related kinases 1/2 (ERK1/2)]. Like RAFTK phosphorylation, ERK1/2 activation is PTX sensitive and can be attenuated by the depletion of intracellular Ca 2ϩ and by PKC inhibition, suggesting that RAFTK might mediate the glutamatedependent activation of ERK1/2. Furthermore, we demonstrated that glutamate stimulation of primary astrocytes leads to a significant increase in DNA synthesis. Glutamate-stimulated DNA synthesis is PTX sensitive and can be inhibited by the MAP kinase kinase inhibitor PD98059, suggesting that in primary astrocytes, glutamate might signal via RAFTK and MAP kinase to promote DNA synthesis and cell proliferation. Key Words: Glutamate -Primary astrocytes-Mitogen-activated protein kinase -Tyrosine kinase -Related adhesion focal tyrosine kinase (RAFTK).