Characterization of human copine III as a phosphoprotein with associated kinase activity.

The copines, first described by Creutz et al. [(1998) J. Biol. Chem. 273, 1393-1402], comprise a two C2 domain-containing protein family and are known to aggregate phosphatidylserine membranes in a calcium-dependent manner. No enzymatic function has been attributed to copines yet. Due to a cross-reacting activity of Mikbeta1, an antibody to the IL-2Rbeta chain, we were able to serendipitously purify, partially microsequence, and clone human copine III. The 5 kb copine III transcript is expressed ubiquitously as determined by a multitissue Northern blot analysis. Phosphoamino acid analysis revealed phosphorylation of copine III on serine and threonine residues. In vitro kinase assays were performed with immunoprecipitated endogenous copine III, chromatography-purified endogenous copine III, and recombinant copine III expressed in Saccharomyces cerevisiae. The exogenous substrate myelin basic protein was phosphorylated in all in vitro kinase assays containing copine III immunoprecipitate or purified copine III. A 60-kDa band was observed in corresponding in gel kinase assays with staurosporine-activated cells. Cell lines expressing high levels of copine III protein had correspondingly high kinase activity in copine III antiserum immunoprecipitate. However, the copine amino acid sequences lack the traditional kinase catalytic domain. Therefore, the data suggest copine III may possess an intrinsic kinase activity and represent a novel unconventional kinase family.

IL-2 activation of NK cells: involvement of MKK1/2/ERK but not p38 kinase pathway.

IL-2 stimulates extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in various immune cell populations. The functional roles that these kinases play are still unclear. In this study, we examined whether MAPK kinase (MKK)/ERK and p38 MAPK pathways are necessary for IL-2 to activate NK cells. Using freshly isolated human NK cells, we established that an intact MKK/ERK pathway is necessary for IL-2 to activate NK cells to express at least four known biological responses: LAK generation, IFN-gamma secretion, and CD25 and CD69 expression. IL-2 induced ERK activation within 5 min. Treatment of NK cells with a specific inhibitor of MKK1/2, PD98059, during the IL-2 stimulation blocked in a dose-dependent manner each of four sequelae, with inhibition of lymphokine-activated killing induction being least sensitive to MKK/ERK pathway blockade. Activation of p38 MAPK by IL-2 was not detected in NK cells. In contrast to what was observed by others in T lymphocytes, SB203850, a specific inhibitor of p38 MAPK, did not inhibit IL-2-activated NK functions. This data indicate that p38 MAPK activation was not required for IL-2 to activate NK cells for the four functions examined. These results reveal selective signaling differences between NK cells and T lymphocytes; in NK cells, the MKK/ERK pathway and not p38 MAPK plays a critical positive regulatory role during activation by IL-2.

Therapeutic targeting of Src-kinase Lyn in myeloid leukemic cell growth.

Protein tyrosine kinases play a major role in promoting cell growth, and their activity in solid tumors is well established. Inhibitors of protein tyrosine kinases are now in advanced clinical trials for the treatment of breast and brain cancers. Because Src-related PTK have been shown to be activated in leukemic cell lines, we studied their activation in human myeloid leukemia. Blasts from the majority of patients with acute leukemia showed constitutive activity of the Src kinase Lyn. In contrast, no patient samples showed constitutive activation of Jak2. Genetic and pharmacologic targeting of Lyn was used to determine its contribution to leukemic cell growth. Antisense Lyn oligonucleotide treatment resulted in the inhibition of tritiated thymidine incorporation following GM-CSF stimulation of the factor-dependent line MO7e. The Src kinase inhibitor PD166285 inhibited the growth of human leukemic cell lines and leukemic blasts. When combined with doxorubicin, an additive effect on the inhibition of leukemic cell growth occurred. These studies demonstrate the importance of Src kinases in promoting leukemic cell growth and suggests that further development of agents which target Src kinases and their inclusion in multidrug regimens are warranted for novel therapies of myeloid leukemia.