Karnitz, Larry M.

Although massive genome sequencing efforts have identified the protein kinases encoded by several eukaryotic genomes and proteomic analyses have begun to determine the kinases expressed in a cell, there is still much to learn about the additional cellular events that shape eukaryotic kinomes. Large-scale analyses in Saccharomyces cerevisiae have indicated that a relatively small subset of kinases requires chaperoning by heat shock protein 90 (Hsp90). However, new evidence suggests that most kinases do require chaperoning and, furthermore, that Cdc37, a chaperone that has Hsp90-dependent and -independent functions, serves as the chaperone for a large portion of the yeast kinome.

Previous studies demonstrated that ataxia telangiectasia mutated– and Rad3-related (ATR) kinase and its downstream target checkpoint kinase 1 (Chk1) facilitate survival of cells treated with nucleoside analogs and other replication inhibitors. Recent results also demonstrated that Chk1 is depleted when cells are treated with heat shock protein 90 (Hsp90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). The present study examined the effects of 17-AAG and its major metabolite, 17-aminogeldanamycin (17-AG), on Chk1 levels and cellular responses to cytarabine in human acute myelogenous leukemia (AML) cell lines and clinical isolates. Cytarabine, at concentrations as low as 30 nM, caused activating phosphorylation of Chk1, loss of the phosphatase Cdc25A, and S-phase slowing. Conversely, treatment with 100 to 300 nM 17-AAG for 24 hours caused Chk1 depletion that was accompanied by diminished cytarabine-induced S-phase accumulation, decreased Cdc25A degradation, and enhanced cytotoxicity as measured by inhibition of colony formation and induction of apoptosis. Additional studies demonstrated that small inhibitory RNA (siRNA) depletion of Chk1 also sensitized cells to cytarabine, whereas disruption of the phosphatidylinositol 3-kinase (PI3k) signaling pathway, which is also blocked by Hsp90 inhibition, did not. Collectively, these results suggest that treatment with 17-AAG might represent a means of reversing checkpoint-mediated cytarabine resistance in AML.

Although diverse signaling events are initiated by stimulation of multichain immune recognition receptors on lymphocytes, it remains unclear as to which specific signal transduction pathways are functionally linked to granule exocytosis and cellular cytotoxicity. In the case of natural killer (NK) cells, it has been presumed that the rapid activation of protein kinase C (PKC) enables them to mediate antibody-dependent cellular cytotoxicity (ADCC) and "natural" cytotoxicity toward tumor cells. However, using cloned human NK cells, we determined here that Fc receptor stimulation triggers granule release and ADCC through a PKC-independent pathway. Specifically, pretreatment of NK cells with the selective PKC inhibitor, GF109203X (using concentrations that fully blocked phorbol myristate acetate/ionomycin-induced secretion) had no effect on FcR-initiated granule release or ADCC. In contrast, FcR ligation led to the rapid activation of phosphatidylinositol 3-kinase (PI 3-kinase), and inhibition of this enzyme with the selective inhibitor, wortmannin, blocked FcR-induced granule release and ADCC. Additional experiments showed that, whereas FcR-initiated killing was wortmannin sensitive and GF109203X insensitive, natural cytotoxic activity toward the tumor cell line K562 was wortmannin insensitive and GF109203X sensitive. Taken together, these results suggest that: (a) PI 3-kinase activation induced by FcR ligation is functionally coupled to granule exocytosis and ADCC; and (b) the signaling pathways involved in ADCC vs natural cytotoxicity are distinct.

The proliferation of antigen-activated T cells is mediated by the T cell-derived growth factor, interleukin 2 (IL-2). The biochemical signaling cascades initiating IL-2-induced growth are dependent upon protein tyrosine kinase (PTK) activity. One IL-2-regulated PTK implicated in this cascade is the Src-family kinase, Fyn. Previous studies have described a physical association between Fyn and a potential downstream substrate, phosphatidylinositol 3-kinase (PI3-kinase) as well as the IL-2-dependent activation of PI3-kinase in T cells; however, the role of Fyn in IL-2-induced PI3-kinase activation remains unclear. In this report, we demonstrate that IL-2 stimulation triggers tyrosine phosphorylation of the p85 subunit of PI3-kinase in the murine T cell line, CTLL-2. Lysates prepared from growth factor-deprived and IL-2-stimulated T cells reconstituted both the binding of CTLL-2 cell-derived Fyn to and the IL-2-inducible tyrosine phosphorylation of exogenously added recombinant p85. Furthermore, overexpression of wild-type Fyn in these cells enhanced both the basal and IL-2-mediated activation of PI3-kinase. Additional studies of the Fyn-PI3-kinase interaction demonstrated that the Src homology 3 (SH3) domain of Fyn constitutes a direct binding site for the p85 subunit of PI3-kinase. These results support the notion that Fyn may be directly involved in the activation of the downstream signaling enzyme, PI3-kinase, in IL-2-stimulated T cells.

Crosslinking of the low affinity immunoglobulin G (IgG) Fc receptor (Fc gamma R type III) on natural killer (NK) cells initiates antibody-dependent cellular cytotoxicity. During this process, Fc gamma R stimulation results in the rapid activation of phospholipase C (PLC), which hydrolyzes membrane phosphoinositides, generating inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol as second messengers. We have recently reported that PLC activation after Fc gamma R stimulation can be inhibited by a protein tyrosine kinase (PTK) inhibitor. Based on the paradigm provided by the receptor tyrosine kinases, we investigated whether PLC-gamma 1 and/or PLC-gamma 2 are expressed in NK cells, and whether the PLC-gamma isoforms are tyrosine phosphorylated in response to Fc gamma R stimulation. Immunoblotting analyses with PLC-gamma 1- and PLC-gamma 2-specific antisera demonstrate that both isoforms are expressed in human NK cells. Furthermore, Fc gamma R crosslinking triggers the tyrosine phosphorylation of both PLC-gamma 1 and PLC-gamma 2 in these cells. Phosphorylation of both isoforms is detectable within 1 min, and returns to basal level within 30 min. Pretreatment with herbimycin A, a PTK inhibitor, blocked the Fc gamma R-induced tyrosine phosphorylation of PLC-gamma 1 and PLC-gamma 2, and the subsequent release of inositol phosphates. These results suggest that Fc gamma R-initiated phosphoinositide turnover in human NK cells is regulated by the tyrosine phosphorylation of PLC-gamma. More broadly, these observations demonstrate that nonreceptor PTK(s) activated by crosslinkage of a multisubunit receptor can phosphorylate both PLC-gamma isoforms.