Oncostatin-M (OSM) is a potent mitogen for Kaposi's sarcoma (KS) cells. We studied signaling by the OSM receptor in three AIDS-related KS lines and show induction of tyrosine phosphorylation of 145-, 120-, 85-, and 42-kD substrates. The 42-kD substrate was identified as p42MAPK (mitogen-activated protein kinase), also known as ERK-2. This serine/threonine kinase relays mitogenic signals from receptor tyrosine protein kinases (TPKs) or receptor-associated TPKs to transcriptional activators. The OSM dose dependence for MAP kinase activation and induction of KS cell growth were almost identical, suggesting functional linkage. MAP kinase activation was dependent on tyrosine phosphorylation, and both OSM-induced MAP kinase activity and KS cell growth could be suppressed by TPK inhibitors, genistein and geldanomycin. OSM also stimulated tyrosine phosphorylation of similar substrates and MAP kinase activity in human vein endothelial cells. While it has been proposed that the OSM receptor may include the gpl30 subunit of the IL-6 receptor and a-chain of leukemia inhibitory factor (LIF) receptor, neither LIF nor r.IL-6 induced tyrosine protein phosphorylation or p42MI'K activation in KS cells.However, r.IL-6 did stimulate tyrosine phosphorylation and p42'PK activity in the human B cell line, AF-10, while OSM and LIF exerted no effects. Our results indicate that, although the OSM and IL-6 receptors share a common signaling pathway, this pathway is selectively activated by OSM in Kaposi's cells. (J. Clin. Invest. 1993. 92:848-857.)

Ligation of membrane immunoglobulin M (mIgM) receptor in the Ramos B-cell line induced tyrosine phosphorylation of several intracellular substrates, including the adaptor protein. Shc. Phosphorylated Shc could be seen to associate with Grb2 in a complex which included hSOS. Inasmuch as hSOS is involved in p21ras activation, we also demonstrated that mIgM ligation activated a Ras-dependent kinase cascade in which sequential activation of Raf-1 and MEK-1 culminates in the activation of p42 mitogen-activated protein (MAP) kinase (ERK-2). The tumour promoter and protein kinase C agonist, phorbol 12-myristate 13-acetate (PMA), also activated Raf-1, MEK-1, and MAP kinase in Ramos cells, but did not induce tyrosine phosphorylation of Shc or Shc/Grb2 association. Okadaic acid, another tumour promoter and serine/threonine phosphatase inhibitor, activated p42 MAP kinase without activating Raf-1 or MEK-1, suggesting the existence of a serine/threonine phosphatase which directly regulates MAP kinase activity.

No abstract

JK32.1 and SKW6.4 are Epstein-Barr virus (EBV)-positive human B cell lines that undergo spontaneous, lymphocyte function-associated antigen 1 (LFA-1) dependent homotypic adhesion in culture. This process is associated with induction of tyrosine phosphoproteins of molecular mass 90, 106, and 120 kDa and could be reproduced when these cells were centrifugationally aggregated. Antibodies to the beta 2 (CD18) chain of LFA-1 interfered with induction of p120, p106, and p90 during cellular aggregation. Response induction was abrogated when cells were incubated with protein tyrosine kinase (PTK) inhibitors (erbstatin, genistein, and geldanomycin) or cytochalasin B prior to aggregation. An in vitro kinase assay did not reveal activation of focal adhesion kinase. Although the role of LFA-1-dependent tyrosine phosphorylation in B cells is uncertain, patients with the leukocyte adhesion defect (LAD) exhibit humoral abnormalities. Moreover, aggregation did not induce specific tyrosine phosphoproteins in an EBV-transformed B cell line from a LAD patient. These results suggest that an LFA-1-dependent PTK pathway may play an important role in human B cell function.

IL-6 is a multi-functional cytokine that utilizes 80-kDa ligand-binding and 130-kDa signal-transducing subunits to stimulate diverse cellular responses. Although IL-6R ligation has been associated with tyrosine protein phosphorylation and activation of an unidentified serine/threonine kinase, very little is known about the intermediary signaling events between the cell membrane and the nucleus. rIL-6 treatment of the human B cell line, AF-10, induced MAP kinase (mitogen-activated protein kinase) activity as determined by in vitro phosphorylation of microtubule-associated protein-2 (MAP-2) and the synthetic peptide APRTPGGRR, corresponding to amino acids 95-98 of bovine myelin basic protein. The kinetics of the response was rapid and dependent on the dose of rIL-6. The response was cytokine specific, did not require the presence of extracellular Ca2+, and was minimally affected by the presence of staurosporine. MAP kinase activation in AF-10 cells occurred in parallel with appearance of 42- and 44-kDa tyrosine phosphoproteins (p42 and p44). Moreover, MAP kinase activation was diminished when AF-10 cells were stimulated with rIL-6 in the presence of tyrosine protein kinase inhibitors, genistein and geldanomycin. p42 and p44 co-electrophoresed on SDS-PAGE with extracellular signal-related kinase (ERK)-2, and ERK-1, respectively; both are members of the ERK family. In addition to p42MAPK and p44MAPK, rIL-6 also activated a MAP-2 kinase that eluted at a lower salt concentration (20 to 60 mM NaCl, peak I) from Mono-Q resin than p42MAPK (120 to 180 mM NaCl, peak II). The identify of this kinase is unknown but it is not an MPB kinase or a protein that exhibits immunoreactivity with anti-ERK antisera. In another IL-6-responsive B cell line, SKW6.4, rIL-6-activated peak I MAP-2 kinase but failed to activate ERK-2. The protein kinase C agonist, PMA, did, however, activate ERK-2 in SKW6.4 cells. These results show that the pleiotrophic cytokine, IL-6, activates p42MAPK/ERK-2 and at least one other serine/threonine kinase in B cell lines.