MINI-REVIEW and MEETING REPORT Serine/Threonine

Home , Binding site, Protein kinase, Protein Kinase C

Serine/threonine phosphorylation in cytokine signal transduction JA McCubrey1,2, W Stratford May3, V Duronio4 and A Mufson5

1Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC; 2Member Leo Jenkins Cancer Center, East Carolina University; 3Sealy Center for Oncology/Hematology, UT Medical Branch at Galveston, Galveston, TX, USA; 4Dept of Medicine, University of British Columbia and VH&HSC, Jack Bell Research Centre, Vancouver, BC, Canada; and 5Cancer Immunology and Hematology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

Over the past decade, the involvement of tyrosine kinases in which included a cytokine/interferon receptor with associated signal transduction pathways evoked by cytokines has been Jak kinases and downstream STAT transcription factors. The intensively investigated. Only relatively recently have the roles of serine/threonine kinases in cytokine-induced signal trans- Jak kinases would be activated upon ligand-induced receptor duction and anti-apoptotic pathways been examined. Cytokine oligomerization. They would phosphorylate STAT molecules receptors without intrinsic kinase activity such as interleukin-3 on tyrosine residues, which would allow the STAT molecules (IL-3), granulocyte–macrophage colony-stimulating factor (GM- to dimerize, promote nuclear translocation, increase their CSF) and the interferons were thought to transmit their regulat- DNA binding activity and regulate gene expression. Many ory signals primarily by the receptor-associated Jak family of cytokine receptors were shown to utilize this basic signal tyrosine kinases. This family of tyrosine kinases activates STAT transcription factors, which subsequently transduced transduction model (eg IL-2R, IL-3R, IL-5R, Epo-R, GM-CSFR their signals into the nucleus to modulate gene expression. and others). As with most hypotheses, however, this simple Cytokine receptors with intrinsic tyrosine kinase activity such model was incomplete, and ignored the branched nodal as c-Kit were initially thought to transduce their signals inde- nature of intracellular signaling cascades. pendently of serine/threonine kinase cascades. Recently, both Studies of the stem cell factor (SCF) induced biological of these types of receptor signaling pathways have been shown activity illustrate well the downstream complexity and to interact with serine/threonine kinase pathways as maximal activation of these tyrosine kinase regulated cascades involve requirement for serine/threonine phosphorylation to integrate serine/threonine phosphorylation modulated by, for example cytokine-induced tyrosine phosphorylation with the down- MAP kinases. A common intermediate pathway initiating from stream signaling cascades regulating differentiation and cell cytokine receptors is the Ras/Raf/MEK/ERK (MAPK) cascade, proliferation. SCF can be synthesized as membrane-bound or which can result in the phosphorylation and activation of soluble forms. However, the differences these two forms have additional downstream kinases and transcription factors such on signal transduction and biological responses are not well as p90Rsk, CREB, Elk and Egr-1. Serine/threonine phosphorylation is also involved in the regulation of the apoptosis-con- understood. SCF transduces its effects through the c-Kit recep- trolling Bcl-2 protein, as certain phosphorylation events tor, which has intrinsic tyrosine kinase activity. Receptors induced by cytokines such as IL-3 are anti-apoptotic, whereas such as c-Kit which have tyrosine kinase activity recruit sig- other phosphorylation events triggered by chemotherapeutic naling molecules which bind the autophosphorylated recep- drugs such as Paclitaxel are associated with cell death. tors or their tyrosine phosphorylated substrates. This binding Serine/threonine phosphorylation is implicated in the etiology occurs via a Src homology 2 domain (SH2, a protein domain of certain human cancers as constitutive serine phosphorylation of STATs 1 and 3 is observed in chronic lymphocytic leu- related to phosphotyrosine binding domain of Src). kemia and can be inhibited by the chemotherapeutic drug flu- Dr David Williams (Howard Hughes Medical Institute, Indi- darabine. Serine/threonine phosphorylation also plays a role in ana University, Indianapolis, IN, USA) has used specific the etiology of immunodeficiencies. Activated STAT5 proteins knockout and transgenic mice to examine the differential are detected in reduced levels in lymphocytes recovered from ability of membrane-bound and soluble forms of SCF to HIV-infected individuals and immunocompromised mice. induce downstream signal pathways.7,8 Dr Williams’ work Serine/threonine phosphorylation may be an important target indicates that activation of extracellular regulated kinases 1 of certain chemotherapeutic drugs which recognize the acti- MAPK MAPK vated proteins. This meeting report and mini-review will dis- and 2 (ERK1 and ERK2, also known as p42 and p44 ), cuss the interactions of serine/threonine kinases with signal p38MAPK and phosphatidylinositol 3-kinase (PI3K) occurs in transduction and apoptotic molecules and how some of these response to bone marrow-derived stromal cells expressing the pathways can be controlled by chemotherapeutic drugs. Leuke- membrane-restricted but not the soluble form of SCF.8 More- mia (2000) 14, 9–21. over, differences were also observed in the development of Keywords: signal transduction; phosphorylation; cytokine; apoptosis; chemotherapeutic drugs the two different types of mice. Introduction of the membrane- restricted form of murine SCF into Steel-Dickie mice, which contain a mutation in the gene encoding SCF, resulted in a significant improvement in the production of red blood cells Introduction and a decrease in bone marrow hypoplasia and runting.2 However, when the full-length soluble form of SCF was intro- In the early 1990s, the identification of the Jak tyrosine kinases duced into these mutant mice, no improvement was observed. and their roles in cytokine signal transduction were eluci- Thus the proliferative and differentiative effects induced by dated.1–6 A relatively simple signal transduction model arose SCF may involve tyrosine and serine/threonine kinases. These data demonstrate that, the two different forms of SCF have distinct biological functions. An overview of some of the Correspondence: JA McCubrey, Department of Microbiology and Immunology, East Carolina University School of Medicine, Green- effects of cytokines on signal transduction pathways is ville, NC 27858, USA; Fax: 252 816 3104 presented in Figure 1. In the following discussion, we will Received 22 September 1999; accepted 4 October 1999 consider the role of the most important serine/threonine kin- Review JA McCubrey et al 10

Figure 1 Cytokine regulation of signal transduction and apoptotic molecules. The effects of cytokines and interferons on signal transduction and apoptotic molecules are indicated. Some of the effects and sites of serine/threonine and tyrosine kinases are highlighted in yellow. A red ‘P’ in a white circle indicates phosphorylated serine/threonine residues on proteins. A black ‘P’ in a white circle indicates phosphorylated tyrosine residues. Cytokine receptors are shown in black, cytokines are shown in red. Solid lines indicate the pathways initiated after receptor ligation. The intermediary signal transducing molecules are indicated in orange. The kinases are indicated in green. Transcription factors are indicated in blue. Pro-apoptotic proteins are indicated in brown, as is the Shp2 phosphatase. Anti-apoptotic molecules are indicated in maroon. Chaperonin molecules are indicated in black. Phospholipid (PL) and diacyl glycerol (DAG) are indicated in orange. Skull and cross bones indicates that apoptosis is induced. Skull and cross bones with a red ‘X’ through it indicates that apoptosis is prevented.

ases that participate in cytokine signaling and the biological PI3K is a heterodimeric protein consisting of an 85-kDa consequences of their activation and dysregulation. regulatory subunit, which contains SH2, and SH3 domains (Src homology region 3, a protein domain related to a proline rich binding domain found in Src), and a 110 kDa catalytic Phosphatidylinositol 3-kinase (PI3K) and Akt/protein kinase subunit.9–17 PI3K belongs to a multi-gene family, which has B (PKB) been divided into three classes based on their lipid substrate speciﬁcities, the presence of certain key structural motifs and The stimulation of appropriate target cells by cytokines also perhaps by the mechanisms by which the proteins are regu- leads to the rapid activation of PI3K.9–14 P13Ks can catalyze lated. There are three different isoforms of p110 expressed in the conversion of phosphatidylinositol (PtdIns), PtdIns(4)P hematopoietic cells (␣, ␤, ␦) with the ␦ isoform appearing to 9–13 and PtdIns(4,5)P2 into PtdIns(3)P, PtdIns(3,4)P2 and be restricted to only hematopoietic cells. However, the 14 PtdIns(3,4,5)P3. The lipid products of PI3K serve to localize roles that these different isoforms have in cytokine signaling and activate additional downstream signal transduction mol- remain to be fully elucidated. IL-3-induced tyrosine phos- ecules (Rac, protein kinase B (PKB also known as Akt)) in the phorylation of the IL-3 receptor ␤-chain creates a docking site vicinity of the cell membrane. These PI3K lipid products vary at tyrosine 612 (Y612) for the tyrosine phosphatase SHP2.10 in terms of their abundancy in cells. For example, PtdIns(3)P SHP2 can then associate with the adaptor protein, which sub- is always present in cells and does not increase upon cellular sequently interacts with the SH2 domain of the p85 subunit 10 stimulation. In contrast, the levels of PtdIns(3,4) P2 and of PI3K. The adaptor protein can also bind phosphorylated PtdIns(3,4,5) P3 are present only in very low levels in resting tyrosine residues on Src family kinases, linking PI3K catalytic cells and increase dramatically upon stimulation.14 subunits to tyrosine kinase regulated signal transduction path-

Leukemia Review JA McCubrey et al 11 ways.14 This may serve to localize PI3K to the cellular mem- significant PKB activation, and utilized dominant negative brane, which is rich in lipid substrates. In this context, PI3Ks forms of p85 to show the lack of involvement of the PI3K may be able to interact with the Ras GDP/GTP exchange pro- pathway in phosphorylation of Bad. In some cases, phos- teins in a GTP-dependent fashion. Ras has been shown to phorylation of Bad that was dependent upon PI3K could be function either upstream or downstream of PI3Ks depending observed, which suggests that this effect may be cell type upon the particular stimulus.14 dependent. In fact, it is possible that Bad phosphorylation at Once the p85 PI3K subunit is phosphorylated, activation of S112 could occur in a PI3K-dependent fashion if the PI3K the p110 catalytic subunit occurs that in turn activates the pathway could contribute to activation of the MAPK pathway, downstream targets p70 ribosomal S6 kinase (p70S6K), PKB, as has been suggested from some studies.27 Dr Duronio dis- also known as Akt, and NF-␬B.18–21 The drugs wortmannin cussed results of studies showing that in MC/9 cells, MAPK and Ly 294002 are specific inhibitors of PI3K. In addition, activation was not dependent upon PI3K activation.22 The there also exist dominant negative mutants of p85 and anti- main argument put forward was that while wortmannin could sense vectors. Rapamycin is an inhibitor of p70 S6 kinase acti- partially block activation of MAPK, as has been reported in vation.15 Rapamycin inhibits p70S6 kinase by a protein numerous studies, concentrations of another PI3K inhibitor, referred to as target of rapamycin (TOR). These reagents allow Ly294002, that completely block PI3K had no effect on MAPK the investigator the ability to determine the importance of this activation. In another recent study,28 dominant negative forms pathway in different physiological conditions. of PI3K were used to show that selective inhibition of Raf iso- PKB is also a member of a multigene family. Currently three forms may explain the ability of the PI3K pathway to affect members of this family have been identified. The PKB family MAPK in some cell systems, but not others. In summary, a members all contain an amino-terminal pleckstrin homology variety of cytokines has been shown to activate the P13K path- (PH) domain, a catalytic domain and finally a carboxyl ter- way, but the exact relevance of that pathway, and particularly minal domain which lacks homology with other proteins.14,16 the phosphorylation of Bad, to cell survival may depend on Activation of PKB occurs in two phases, phosphorylation on the cellular context, as seems to be the case for interaction of serine/threonine residues and targeting to the lipid rich cell the PI3K and MAPK pathways. membrane by the N-terminal PH domain.9 The PH domain Another aspect of PKB regulation also involves dephos- may mediate binding to certain lipid activators. Phosphoryl- phorylation of PI3K-generated lipids by the SH2-containing ation of threonine 308 (T308 in the catalytic domain) and ser- inositol 5-phosphatase, or SHIP, protein. Studies using knock- ine 473 (S473 in the carboxyl-terminal domain) results in the out mice lacking SHIP were done by the laboratories of activation of PKB. T308 is phosphorylated by the PtdIns(3,4) Duronio and Krystal, whose laboratory was one of several that 29 P2(3,4,5)P3-dependent kinase (PDK1). The kinase which phos- first cloned SHIP. In bone marrow-derived mast cells from phorylates S473 is currently unknown, but is referred to as SHIP−/− mice, steel factor was found to cause a tremendous

PDK2. Some evidence suggests that PDK2 may be the same accumulation of PI(3,4,5)P3, that was as much as 100-fold as the integrin linked kinase (ILK).17 High levels of PKB activity higher than that in the SHIP+/+ cells,30 while under the same −/− are only observed when both T308 and S473 are phosphoryl- conditions the SHIP cells had lower levels of PI(3,4)P2. ated.9,18 These results show that SHIP is the primary 5-phosphatase in The pro-apoptotic protein Bad may play an important role hematopoietic cells. More recent studies were discussed that in determining whether a cell undergoes apoptosis. Korsmeyer demonstrate the importance of both the PI(3,4,5)P3 and and colleagues showed that phosphorylation of Bad at S112 PI(3,4)P2 lipids in regulation of PKB activity and phosphoryl- and S136 resulted in its association with the cytosolic chap- ation of PKB at the PDK1 and PDK2-dependent sites. eronin protein 14-3-3, thereby sequestering it away from its 19 binding partner Bcl-XL at the mitochondria. Subsequent reports have suggested that one role of PI3K and PKB in pre- Raf/MEK/ERK activation by interferons, abrogation of venting apoptosis is due to phosphorylation of Bad by PKB at cytokine dependency and prevention of apoptosis by S136.20,21 Dr Vincent Duronio (University of British Columbia, activated Raf and MEK oncoproteins Vancouver, BC, Canada) discussed the state of phosphorylation of Bad in response to cytokines, and showed that phos- Dr Andrew Larner (Cleveland Clinic Foundation, Cleveland, phorylation of Bad could occur independently of PKB acti- OH, USA) presented evidence indicating that in addition to vation.18 One explanation for the discrepancy is that the latter being tyrosine phosphorylated, several STATs (STAT1␣, study examined phosphorylation of endogenous Bad protein STAT3 and STAT4) are phosphorylated on a conserved serine by the endogenous PKB. Previous work examined tagged and residue.31 This serine residue is a target of the serine/threonine overexpressed Bad, which was found to be phosphorylated by kinase ERK. INF␣/␤ and INF␥ can activate Raf-1 and B-Raf overexpressed PKB. Furthermore, in mouse MC/9 cells, Bad which will in turn activate MEK and the downstream ERK1 protein was not significantly phosphorylated at S136 in and ERK2 kinases.31–35 Serine phosphorylation of STAT1 and response to cytokines. Evidence was presented that phos- STAT3 is required for these STATs to maximally transactivate phorylation at S112 was mediated by a MEK-dependent path- gene expression. Dr Larner described how ERK is activated in way, and this phosphorylation event was sufficient to regulate response to interferons (IFN). Using embryonic stem cells with 22,23 association of Bad with Bcl-XL. a targeted deletion of B-Raf, Dr Larner was able to elucidate Furthermore, analysis of tryptic peptide maps of the roles for B-Raf in the anti-viral and anti-proliferative effects of endogenously phosphorylated Bad revealed at least one novel INF-␣.34 Furthermore, transfection of Jurkat T cells with a kin- site of phosphorylation distinct from either S112 or S136. The ase-inactive form of B-Raf significantly reduced the anti-pro- relevance of cytokine-dependent PKB phosphorylation as a liferative effects of IFN-␣. These results demonstrate that an key player in prevention of apoptosis by some cytokines has intricate cross-talk occurs between the Jak and Raf kinases in been brought into question by studies from the laboratories of IFN signaling and that the Raf kinase pathway is essential for Duronio18,24 and more recently Welham.25,26 The latter stud- the biological effects of type 1 interferons. ies showed that cell survival could occur in the absence of Additional studies have shown intricate interactions occur

Leukemia Review JA McCubrey et al 12 between tyrosine kinase, serine/threonine kinases and STAT heparin binding epidermal growth factor (hb-EGF).46,47 Raf transcription factors, which can either, induce or repress their was stimulating hb-EGF expression by modulating the activity activities.36–39 It has been known for some time that v-Src of ETS transcription factors.46,47 Raf also could induce the expression can increase the Stat-3 binding activity in certain expression of the cell cycle inhibitory protein p21Cip-1. High cells36 suggesting that there can be interactions between a v- levels of Raf would induce more p21Cip-1 protein which was Src induced downstream activity (possibly ERK) and STAT. correlated with cell cycle arrest.48,49 A-Raf, the weakest Raf Moreover, it is now recognized that the Raf kinase can be oncoprotein, was a poor inducer of p21Cip-1. activated by IFN␥ and oncostatin M.31–34,39 Interestingly, acti- Dr McCubrey and colleagues have shown that the three Raf vation of Raf by these factors requires Jak and STATs, but this oncoproteins have a differential capacity to abrogate the cyto- activation is Ras-independent, reducing the possibility of the kine dependency of human (TF-1) and murine (FDC-P1) hem- involvement of an autocrine loop.32,33,39 An unanswered atopoietic cells when expressed from the ⌬Raf-ER construc- question is whether the physical sites of Raf-1 activation by tions. Both TF-1 and FDC-P1 cell lines require either IL-3 or Jak are the same as those involved in activation by Src family GM-CSF to proliferate. Removal of cytokine results in kinases (Y340 and Y341)? Studies performed by Dr Larner and apoptosis within 1 to 3 days. Some of this work was published colleagues have demonstrated an important link between the in the December 1998 issue of Leukemia.40 Two different Jak and Raf signaling pathways. types of cells were isolated after infection with the activated Recently it was hypothesized that Src and not the Jak kin- Raf constructs. The majority of the cells infected with the acti- ases were responsible for activating STAT in IL-3-induced pro- vated Raf proteins remained cytokine-dependent for growth. liferation.38 Furthermore, certain phosphorylation events on A minority of the cells infected with the ⌬Raf:ER proteins grew STAT3 mediated by ERK2 have been shown to actually repress in response to the activated Raf. This, latter class of Raf- its activity.39 Clearly protein phosphorylation of these tran- responsive cells usually expressed higher levels of the Raf pro- scription factors is a complex area with certain events being teins and downstream MEK1 activity than the cells which activating whereas others are inhibitory. remained cytokine-dependent. Interestingly A-Raf was more Downstream targets of Ras and certain protein kinase C efficient than either Raf-1 or B-Raf in abrogating cytokine (PKC) isoforms are the Raf kinases. The abilities of the different dependency of both cell lines. Studies performed by Larry Kar- Raf kinases to abrogate the cytokine dependency of hemato- nitz at the Mayo clinic (Rochester, MN, USA) have indicated poietic cells and induce downstream signal transduction path- that A-Raf, as opposed to Raf-1, may be the predominant Raf ways were discussed by Dr James McCubrey (East Carolina kinase, that is involved in Raf-mediated signal transduction in University, Greenville, NC, USA).40 Abrogation of cytokine cytokine-dependent murine FDC-P1 cells.28 Alternatively A- dependency is often linked to tumorigenesis.41–43 Raf is a Raf is a poor inducer of p21Cipl, and there may be an intricate member of multi-gene family that consists of Raf-1, A-Raf and balance between growth promotion and cell cycle arrest B-Raf. The roles of the individual Raf kinases in cytokine- promoted by the Raf oncoproteins. mediated signal transduction are beginning to be investigated. Abrogation of cytokine dependency was associated with Raf-1 was the first Raf gene isolated and is fairly ubiquitously autocrine growth factor expression. Cells transformed to cyto- expressed. Raf is a serine/threonine kinase, which has been kine-independence by A-Raf produced the highest levels of implicated as a downstream target of many cytokine recep- autocrine GM-CSF expression.40 The autocrine growth factor tors. Raf can pass the signal initiated from Ras, Src-like tyro- expression in the human hematopoietic cells induced by the sine kinases, PKCs, kinase suppressor of Ras (KSR) and poten- activated Raf expression was significant as it was at sufficient tially other serine/threonine kinases, to MEK1 a dual levels to promote the proliferation of the parental cells. More- specificity kinase (tyrosine and serine/threonine). B-Raf is the over, the autocrine growth factor synthesis in the Raf-respon- strongest Raf kinase in terms of its ability to phosphorylate sive cells was important as neutralizing antibodies to GM-CSF its downstream target MEK1. B-Raf has a more limited tissue inhibited the growth of the ⌬Raf:ER-responsive cells. distribution, but it is expressed in neuronal and some hemato- Activated versions of the downstream MEK1 protein poietic cells, as well as other types of cells. Finally, A-Raf is (⌬MEK1:ER) also abrogate the cytokine dependency of human the weakest Raf kinase in terms of its ability to phosphorylate and murine hematopoietic cells, although they showed a MEK1. A-Raf also has a wide tissue distribution, including lower efficiency than either the ⌬A-Raf:ER or ⌬Raf-1:ER con- hematopoietic cells.40,44 structs. This lower efficiency of abrogation of cytokine depen- There is a hierarchy in the ability of the three Raf oncoprote- dency by ⌬MEK1:ER may be related to the position of MEK1 ins to transform NIH-3T3 cells with B-Raf having the greatest in the signal transduction pathway, as it may not be able to capacity.45–47 Next is Raf-1 and finally A-Raf. B-Raf induced transfer its signal to as many downstream targets as Raf. higher levels of MEK1 activity, Raf-1 moderate levels and the Recently there have been numerous reports suggesting lowest levels of MEK1 activity were observed in A-Raf trans- interactions between Raf and apoptotic regulatory pro- formed cells. Many of these experiments were performed with teins.9,14,18–21 To determine whether Bcl-2 overexpression conditional Raf oncoproteins constructed by Dr Martin could synergize with activated Raf or MEK expression and McMahon (UCSF, San Francisco, CA, USA). The activated Raf increase the recovery of cytokine-dependent cells which oncoproteins were linked to the hormone binding domain of expressed the ⌬Raf:ER or ⌬MEK1:ER oncoproteins, ⌬Raf:ER the human estrogen receptor (⌬Raf:ER)46,47 thus the Raf onco- and ⌬MEK1:ER-infected cells were secondarily infected with proteins could be turned-on by the addition of estrogen (␤- a retrovirus encoding Bcl-2. Bcl-2 overexpression was able to estradiol) to the growth medium. Likewise, they could be synergize with activated Raf or MEK1 increasing the frequency turned-off by the removal of ␤-estradiol from the growth at which cytokine-independent cells could be isolated from medium. Recent experiments have used a mutant estrogen the cytokine-dependent ⌬Raf:ER or ⌬MEK1:ER infected cells. receptor domain which is activated in response to 4-hydroxy- These cells were conditionally transformed as they required tamoxifen and not ␤-estradiol. activation of either the ⌬Raf:ER or ⌬MEK1:ER proteins to pro- Dr McMahon showed that the different Raf oncoproteins liferate. These cells in addition, displayed an altered pattern induced an autocrine growth factor for NIH-3T3 cells, namely of apoptosis as it took longer for the cells to undergo apoptosis

Leukemia Review JA McCubrey et al 13 upon culture in the absence of ␤-estradiol. Furthermore these ecules including receptor-encoded tyrosine kinases, receptor- Bcl-2 and ⌬Raf:ER or ⌬MEK1:ER cells were more resistant to associated tyrosine kinases, hematopoietin family receptors certain drugs indicating that Bcl-2 expression could increase (eg IL-3R, GM-CSF-R, erythropoietin-R), Ras or Vav guanine drug resistance. nucleotide exchange family members, PI3K, or other agents The conditionality of the ⌬Raf:ER and ⌬MEK1:ER constructs which induce cell stress. Rac2 is preferentially expressed in has allowed the investigation of the downstream signal trans- hematopoietic cells and regulates actin cytoskeleton organiza- duction pathways induced by IL-3, Raf and MEK in hematopo- tion.75 Altered Rac expression may contribute significantly to ietic cells. In addition to the Jak/STAT pathway and the neoplastic transformation, as activated forms of Rac will trans- Ras/Raf/MEK/ERK pathway, IL-3 can induce the PI3K-p70S6K form NIH-3T3 cells.74,75 In Rac2 knockout mice, dramatic dif- pathway. Dr McCubrey presented evidence indicating that ferences in cellular adhesion and the ability of hematopoietic both Raf and MEK1 can induce the phosphorylation of the cells to migrate in response to chemoattractants were p70S6 kinase, which lies downstream of PI3K. Thus the Raf observed.76 Thus disruption of this signal transduction cascade pathway can cross-talk with other pathways which have can have profound effects on the homing and chemoattractant growth promoting effects. responses of hematopoietic cells. In addition to the Jak-STAT and Raf/MEK/ERK pathways, several alternative signal transduction cascades are activated by Ras-dependent and independent mechanisms.50–52 Cyto- Protein kinase C in cytokine signaling and oncogenesis kines and mitogens may also activate these pathways which rely heavily on serine/threonine kinases to transduce their The role of one of the neutral lipid activated kinase, PKC, in regulatory signals. These related pathways almost certainly Wnt signaling and colon cancer was discussed by Dr Alan interact with the Raf/MEK/ERK pathway (Figure 1). Ras-depen- Fields (University of Texas Medical Branch, Galveston, TX, dent activation of the MEK1 kinase, MEKK1, a USA). An association between altered PKC expression in 77,78 ␤ serine/threonine kinase, is responsible for activating three colon cancer has been observed. PKC II is an upstream MAPK pathways.50–52 MEKK1 phosphorylates and activates regulator in the Wnt signaling pathway.78 Wnt is a cytokine MEK1 and MEK253,54 and the dual serine/threonine and tyro- related to the fibroblast growth factors and may be regulated 54–59 78 ␤ sine stress/extracellular regulated kinases (SEK). Activation by PKC. Wnt transduces its effects through Frz, Dsh, PKC II, ␤ ␤ ␤ of the SEKs (MKK4 and MKK3/MKK6) results in the activation APC and -catenin. Normally PKC II keeps -catenin in low ␤ ␤ of the stress activated protein kinases (SAPKs), JNK and levels. However, in PKC II-transgenic mice, the levels of - p38MAPK.54–59 JNK is a 46-kDa MAP kinase responsible for catenin are increased. These mice have a higher incidence of activating the AP-1 transcription factor component c-Jun by colon cancer. There may also be an autocrine component to phosphorylating its serine residues 63 and 73.58 p38MAPK is this model of colon cancer as one of the target genes activated responsible for phosphorylating and activating certain mem- by this pathway may be Wnt. This experimental model for ␤ bers of the AP-1 transcription factor family, the C/EBP tran- colon cancer has relevance in human cancer, as PKC II is a scription factor family member, CHOP, and the MAPK-asso- target of certain lipids. Thus certain diets rich in these lipids 55,56,60 ␤ 79,80 ciated protein kinase (MAPK-AP). Activation of MEKK1 may serve to activate PKC II and result in colon cancer. preferentially leads to the activation of JNK Ͼ The roles of some of the other PKC isoforms in signal trans- p38MAPK Ͼ ERK1 and ERK2.55 On the other hand, Raf-1 leads duction and apoptotic pathways were discussed by Dr Allan to the activation of ERK1 and ERK2 but does not result in acti- Mufson (NCI, Bethesda, MD, USA).81–85 The tyrosine kinase vated JNK or p38MAPK.61 Although pharmaceutical companies inhibitor genistein blocks both the IL-3 suppression of have produced specific inhibitors to p38MAPK, direct inhibitors apoptosis and Bcl-2 expression.83,84 Moreover, D609, a spe- to JNK and ERK have not yet been developed. However, the cific inhibitor of phosphatidylcholine-specific phosopholipase ERKs can be indirectly inhibited using compounds such as C, also inhibits IL-3-mediated Bcl-2 expression without affect- PD98059. ing IL-3-induced tyrosine phosphorylation.84 Treatment of Recent evidence suggests that a significant amount of cross- human cytokine-dependent erthroleukemic TF-1 cells with talk occurs among the PI3K, MEKK/SEK/SAPK and D609 induces apoptosis even in the presence of IL-3. Dr Muf- Raf/MEK/ERK pathways.62–66 PI3K has been implicated in son has hypothesized that these data indicate that there is a MEKK1 activation, as well as MEK1/ERK activation,62–66 bifurcation in the signaling pathways downstream of IL-3 whereas oncogenic Raf-1 and MEK1 have also been reported induced tyrosine phosphorylation. Since IL-3 and GM-CSF to activate p70S6K in a PI3K-independent way.66 Inhibitors of stimulate increases in diacylglycerol without mobilizing intra- the PI3K-p70S6K pathway, Wortmannin and rapamycin, exert cellular Ca++, Dr Mufson hypothesized that one of the novel their effects at different points in the pathway.67–72 Wortman- Ca++-independent isoforms of PKC was involved in the sup- nin inhibits PI3K, while the immunosuppressive drug rapamy- pression of apoptosis in IL-3/GM-CSF-dependent hematopo- cin inhibits p70S6K via an upstream target of rapamycin (TOR) ietic cells. To test this hypothesis, Dr Mufson transfected TF- (Figure 1). However, once cells have entered the cell cycle, 1 cells with expression constructs containing PKC␦ or PKC⑀.85 inhibition of p70S6K does not stop proliferation.64,72 Use of Overexpression of PKC⑀ in TF-1 cells had a survival effect and these inhibitors has documented the PI3K-independent acti- prevented apoptosis in TF-1 cells. In contrast, overexpression vation of p70S6K.72 Rapamycin has been recognized for years of PKC␦ did not have an anti-apoptotic effect. PKC⑀ transfec- as a potentially useful immunosuppressive drug.73 Thus these tants remained viable after 72 to 96 h of cytokine deprivation, inhibitors, or more likely derivatives of them, may be useful and the cells were distributed in all phases of the cell cycle, in the treatment of patients with certain leukemias. while little cellular DNA was detected in vector-alone or Dr Williams also discussed some recent data concerning a PKC␦-transfected cells.85 Rac2 knockout mouse model. Rac2 is a member of the Rac Dr Mufson has shown that downregulation of PKC by a 24- family of GTPases, which is involved in coupling signals from h incubation in medium containing 100 nM phorbol-12-myr- membrane receptors to downstream kinase cascades.74–76 Rac istate-13-acetate (PMA) in the presence of IL-3 dramatically molecules can be activated by many signal transducing mol- reduced Bcl-2 levels and induced apoptosis.83 Inhibition of

Leukemia Review JA McCubrey et al 14 PKC by treatment with the PKC inhibitor H7, suppressed the CLL.100 Fludarabine inhibits IFN␣-induced STAT1 activation induction of Bcl-2 mRNA in factor-deprived TF-1 cells stimu- and decreases STAT1 mRNA levels. Eighty to 90% of CLL lated with IL-3. Studies with the mRNA transcription inhibitor patients respond to fludarabine, but the response is not pro- actinomycin D indicated that transcription plays a major role longed and the treatment must be repeated. Furthermore, the in maintaining Bcl-2 levels in TF-1 cells. Dr Mufson therefore loss of STAT1 in CLL patients undergoing fludarabine treat- hypothesized that IL-3 may induce Bcl-2 expression by activ- ment likely results in severe immunodeficiencies as seen in ating PKC. Dr Mufson further examined this hypothesis by STAT-1 deficient mice. Thus there are significant roles of examining the levels of Bcl-2 protein in PKC⑀ and PKC␦ trans- STAT1 in the etiology and potential treatment of certain fected cells.85 PKC⑀ induced Bcl-2 protein expression to levels human leukemias. Identification of the kinases, which phos- significantly greater than those detected in vector-alone or phorylate STAT1 and the downstream targets of STAT1 will PKC␦-transfected cells. Thus overexpression of PKC⑀ may pre- increase our understanding, and may improve the treatment vent apoptosis of hematopoietic cells by increasing Bcl-2 of CLL and other diseases. levels. Dr Robert Kirken (MD Anderson Cancer Center, Houston, TX, USA) discussed the roles of the STAT and JAK proteins in immunodeficiencies. By creating a series of knockout mice, RSK phosphorylation and activation of downstream the roles of the JAK and STAT proteins in immunodeficiencies transcription factors are beginning to be elucidated.101–111 Absence of STAT5a and STAT5b can result in a loss of the ability of certain hematopo- Additional downstream targets of PKC and Raf/MEK/ERK are ietic cells to proliferate in response to certain cytokines.105,106 p90Rsk, CREB, Elk and Egr-1. p90Rsk is a serine/threonine kin- Moreover reduced levels of STAT5a have been detected in ase, while CREB, Elk and Egr-1 are transcription factors. These the spleens and lymph nodes of tumor-challenged mice. Thus molecules have been proposed to be regulated by the ERK STAT5a may have a significant role in tumor induced molecules. GM-CSF treatment of TF-1 cells leads to activation immunosuppression.112 of p90Rsk, CREB phosphorylation and Egr-1 transcription.86– In hematopoietic cells isolated from HIV-infected individ- 92 The regulation of these transcription factors and kinases in uals, the levels of mRNA encoding STAT5a are not affected, response to cytokines was discussed by Dr Kathleen Sakamoto but the levels of STAT5a protein are decreased.113 It is likely (UCLA, Los Angeles, CA, USA).86–92 Dr Sakamoto described that HIV-1 infection alters a post-transcriptional mechanism studies to determine the differential roles of the ERK1 and regulating STAT5a protein levels. Thus part of the immuno- ERK2 proteins in regulation of CREB phosporylation and Egr- deficiency observed in AIDS patients may be due to decreased 1 transcription in response to GM-CSF. Stimulation of TF-1 levels and activities of this STAT protein. cells with GM-CSF results in the phosphorylation of p90Rsk on Dr Kirken has investigated the regulation of STAT5a threonine 359. Treatment of TF-1 cells with the MEK1 inhibi- serine/threonine phosphorylation in a variety of cells.114–116 tor PD98059 results in a decrease in the extent of phosphoryl- Unlike other STAT proteins, STAT5a does not contain a classi- ation of ERK1, ERK2, and pp90Rsk. In addition, transient trans- cal MAPK recognition sequence, and is not phosphorylated fections of TF-1 cells with kinase-defective p90Rsk inhibited by this kinase. Thus, the serine/threonine kinase, which phos- GM-CSF induced transcriptional activation of Egr-1. In con- phorylates STAT5a, remains unknown. The roles of STAT5a trast, transient transfection of TF-1 cells with dominant-nega- phosphorylation in hematopoietic signal transduction are tive ERK1 did not inhibit GM-CSF induced transcriptional acti- being further investigated using phospho-STAT specific anti- vation of Egr-1. Thus Dr Sakamoto proposed that GM-CSF bodies and ‘in-gel’ kinase assays. induces CREB phosphorylation and Egr-1 transcription through an ERK2 but not ERK1 dependent pathway. Although the ERK1 and ERK2 molecules often appear to be co- Regulatory proteins which recognize and bind serine expressed, it is now becoming apparent that they can have threonine phosphorylated proteins–potential for different biological properties. intervention with chemotherapeutic drugs

Some of the proteins involved in signal transduction which Roles of serine phosphorylation of STAT proteins in recognize and bind the serine/threonine phosphorylated pro- leukemia and immunodeficiencies including AIDS teins have been isolated and characterized.117–122 Dr Kun Ping Lu (Harvard University, Boston, MA, USA) described tech- Deregulated serine/threonine phosphorylation of STAT mol- niques to isolate the Pin genes encoding these proteins and ecules is likely involved in diverse pathological conditions discussed how these low molecular weight proteins belong and may provide a target for therapeutic intervention. The to a large multi-gene family. Pin proteins are peptidyl-prolyl recent ability to study STAT phosphorylation in human tumors cis/trans isomerases (PPIase). The Pin1 protein catalyzes the was discussed by Dr David Frank (Dana Farber Cancer Insti- isomerization of phosphorylated Ser/Thr-Pro motifs, which are tute, Boston, MA, USA). The development of a powerful series present on key mitosis-specific phosphoproteins. These pro- of antibodies which specifically recognize the activated STAT teins can inhibit entry into mitosis but are required for cell proteins has allowed Dr Frank to demonstrate that in chronic cycle progression. Pin1 interacts with a subset of mitotic pro- lymphocytic leukemia (CLL), the most common form of leuke- teins, which include Cdc25, Myt1, Wee1, Plk1 and Cdc27. mia in the United States, there is constitive phosphorylation of This group of proteins is also recognized by the mitosis-spe- the STAT1 and STAT3 proteins on serine-727.93–99 In contrast, cific monoclonal antibody MPM-2. The activities of the Pin1 MAPK (ERK1 and ERK2) are not constitutively activated in protein are specific to Ser/Thr-Pro motifs as neither phos- these CLL patients. These observations have clinical signifi- phorylated tyrosine or glutamic acid are able to affect the pro- cance since treatment of CLL patients with fludarabine, leads lyl isomerization. The activities of the Pin1 protein are specific to a specific loss of STAT1. Fludarabine, a purine analogue, to Ser/Thr-Pro motifs as neither phosphorylated tyrosine or is to date the most effective chemotherapeutic agent against glutamic acid are able to affect the prolyl isomerization. Dr

Leukemia Review JA McCubrey et al 15 Lu isolated the Pin genes encoding these proteins and demon- which will mediate the phosphorylation of Bcl-2 at the regu- strated the WW domains to be the first phosphoserine or phos- latory S70 site.127–129 There are also additional serine sites phothreonine-binding modules. Protein-interacting modules contained in the Bcl-2 protein, however by using a series of play a key role in determining the specificity of signal trans- site-directed mutants Dr May and colleagues observed that duction events, and protein phosphorylation is an important alanine substitution for serine at residue S70 represented a loss mechanism in modulating the assembly of specific signaling of survival function phenotype. Thus, an intact S70 site is complexes. WW domains, arrays of conserved 38–40 resi- required for the full and potent anti-apoptotic activity of Bcl-2. dues, are found in a variety of proteins in cell signaling or Staurosporine, a potent kinase inhibitor, can induce regulation, including several proteins involved in cytokine sig- apoptosis by inhibiting PKC-mediated phosphorylation on S70 nal transduction. It has now been demonstrated that WW of Bcl-2. Overexpression of Bcl-2 can inhibit the effects of domains mediate the phosphorylation-dependent protein–pro- staurosporine. These observations suggest that there is a stau- tein interaction by acting as phosphoserine or phosphothreon- rosporine-resistant protein kinase, which also phosphorylates ine-binding modules, with properties analogous to SH2 Bcl-2 on S70. Addition of high concentrations of staurosporine domains, phosphotyrosine-binding modules. Pin1 and related up to 1 ␮M and the MEK1 inhibitor PD98059, led to virtual proteins use their WW domains to interact with a defined sub- complete inhibition of agonist (bryostatin or IL-3) induced Bcl- set of mitosis-specific phosphoproteins which are also recog- 2 phosphorylation at S70 and also additively induced nized by the mitosis-specific monoclonal antibody MPM-2. apoptosis. The possibility that ERK2 was a staurosporine-resist- These Pin1 targets include key mitotic phosphoproteins ant protein kinase was indicated by the observation that ERK2 Cdc25, Myt1, Wee1, Plk1 and Cdc27. Furthermore, Pin1 is and Bcl-2 co-localized to the mitochondria and purified, acti- a peptidyl-prolyl cis/trans isomerase (PPIase), which has an vated ERK2 can phosphorylate Bcl-2 on S70 in a PD98059- extremely high degree of substrate specificity. sensitive, staurosporine-insensitive manner (Figure 2). Further- In contrast to other prolyl isomerases, such as cyclophilins more the non-peptide, polyanionic pharmaceutical drug aur- and FK506-binding proteins, Pin1 specifically isomerizes intricarboxylic acid, which is an activator of ERK, induced phosphorylated Ser/Thr-Pro bonds, whose reaction is normally Bcl-2 phosphorylation in hematopoietic cells deprived of IL- reduced upon phosphorylation. Therefore, Pin1 binds and iso- 3 in a PD98059-sensitive, staurosporine-resistant manner. merizes mitosis-specific phosphoproteins. Importantly, Aurintricarboxylic acid will prolong cell survival when cells inhibiting Pin1 function leads to mitotic arrest and apoptosis. are cultured in the absence of exogenous growth factors (ie Thus, a novel cell cycle regulatory mechanism has been pro- IL-3 and nerve growth factor (NGF). These results indicate that posed, where protein phosphorylation creates binding sites for the serine-threonine kinase ERK2 may be a second physiologi- Pin1, which can then latch on to and isomerize the phos- cal Bcl-2 kinase. Dr May’s group has described a novel mech- phorylated Ser/Thr-Pro peptide bond. In turn, this may change anism by which Bcl-2 can suppress staurosporine-induced the shape of the protein, regulating its dephosphorylation, apoptosis. An overview of some of the effects of degradation or location in the cell. This new post-phosphoryl- serine/threonine kinases on the phosphorylation of apoptotic ation regulatory mechanism may help the cell orchestrate the molecules is presented in Figure 2. complex events of mitosis. The control of apoptosis is a favored target of many thera- Dr Lu has identified and described other related proteins peutic regimes. Thus, many investigators have sought to ident- (Pin 2 and TRF1), which may have roles in connecting mitotic ify drugs, which may modulate Bcl-2 activity.134–140 It has control to the chromosomal telomeric regulatory machin- been shown for some time now that chemotherapeutic ery.121,122 Dysregulation of telomere length has been associa- microtubule destabilizing drugs such as paclitaxel, vincristine, ted with cancer and aging. The mechanisms by which these vinblastin and dolastatin as well as other anti-microtubule Pin proteins recognize the phosphorylated proteins are being agents including colchicine, colcemid and podophyllotoxin investigated by biophysical techniques. This area of research trigger Bcl-2 phosphorylation in certain cells.132–140 Here, may provide potential targets for chemotherapy as some prolyl however Bcl-2 phosphorylation differs from that induced by cis/trans isomerases have already been demonstrated to be the growth factors. First, drug-induced phosphorylation is tem- targets of immunosuppressive drugs (eg cyclosporin, FK506).73 porally slow, requiring at least 2 h to visualize as compared with IL-3. Secondly, phosphorylation is associated with a mor- phologically distinct form of Bcl-2 not observed following IL- Roles of serine/threonine kinases in post-translational 3 or other growth factor treatment of cells. The Bcl-2 phos- modifications of the anti-apoptotic protein Bcl-2 phorylation events associated with these drugs are correlated with cell death. Whether paclitaxel-induced phosphorylat- An intensely investigated area in regulation of cell growth and ation of Bcl-2 inactivates Bcl-2’s anti-apoptotic activity or is prevention of apoptosis is the role of serine/threonine phos- a ‘marker’ of cell death is not yet clear. Moreover, other Bcl- 123–141 phorylation of members of the Bcl-2 family of proteins. 2 family members (Bcl-XL, A1) may also be phosphorylated in For the past few years, the phosphorylation of the anti-apototic response to treatment with paclitaxel, although it should be

Bcl-2 and pro-apoptotic Bad proteins have been examined by noted that Bcl-XL does not appear to be phosphorylated in all many groups. Different phosphoryation events on the Bcl-2 cell types. protein may have antiapoptotic or pro-apoptotic effects. Dr W Dr Subrata Haldar (Case Western Reserve University, Cleve- Stratford May (University of Texas Medical Branch at Galves- land, OH, USA) discussed the effects of paclitaxel and other ton, TX, USA) presented evidence indicating that the drugs on Bcl-2 phosphorylation. Paclitaxel and dolastatin, can serine/threonine kinase ERK2 is a staurosporine-resistant Bcl- trigger Bcl-2 phosphorylation in LnCaP and PC-3 prostate can- 2 kinase (see Figure 2). Stimulation of certain hematopoietic cer cells, the MCF-7 breast cancer line, as well as other cell cell lines with the growth promoting cytokine, IL-3, or the PKC types.135–137 Interestingly, endogenous Bcl-2 phosphorylation activator, bryostatin, will induce the phosphorylation of Bcl- has not been detected in follicular lymphoma cells–the type 2.127–130 These Bcl-2 phosphorylation events are anti-apop- of cells from which the original Bcl-2-IgH translocation was totic in nature. Moreover, PKC is a physiological Bcl-2 kinase, originally identiﬁed. However, since active protein phospha-

Leukemia Review JA McCubrey et al 16

Figure 2 Different effects of Bcl-2 phosphorylation on apoptosis. The effects of Bcl-2 phosphorylation induced by cytokines and microtubule destabilizing drugs are shown. Inactive NF-AT is shown in black, active NF-AT is shown in blue. A red ‘P’ in a white circle indicates phosphorylated serine/threonine residues on proteins. A white ‘P’ with a black circle surrounding it indicates phosphorylation events induced by the microtubule destabilizing drugs. Skull and cross bones indicates that apoptosis is induced. Skull and cross bones with a red ‘X’ through it indicates that apoptosis is prevented.

tase 2A (PP2A) is directly associated with Bcl-2,129 in order to paclitaxel induced Bcl-2 phosphorylation was undetectable. dynamically regulate the protein, an increased level of PP2 Flow cytometry analysis indicated a signiﬁcant reduction in

activity, may explain why phosphorylation has not been the G2/M population due to inhibition of protein synthesis. experimentally observed in these cells. To provide a mechan- This observation reinstates the requirement of the cells to be istic basis for the effect of paclitaxel-induced phosphorylation, cycling to achieve Bcl-2 phosphorylation. However, all-trans Dr Haldar has demonstrated that the mutation of S70 to retinoic acid (ATRA) has also been shown to induce Bcl-2 alanine could not completely abrogate paclitaxel-induced phosphorylation in a pattern different from that of paclitaxel, Bcl-2 phosphorylation138 and that the S87 site in Bcl-2 is also and ATRA will induced differentiation of cells suggesting a necessary for the events induced by these drugs.139 survival role for Bcl-2 phosphorylation by retinoids in myeloid In normal cell cycle progression, Bcl-2 becomes leukemia cells.134

serine/threonine phosphorylated during the G2 to M phase As mentioned earlier by Dr May, there may be several sites transition, the transition phase during which microtubule of serine phosphorylation on the Bcl-2 protein. Okadaic acid, destabilizing drugs are active. In response to treatment with a chemical that inhibits PP2A, as well as other phosphatases,

paclitaxel, cells become arrested in the G2/M transition. Upon stabilizes Bcl-2 phophorylation. Dr Haldar and others have treatment of CLL cells with 5 ␮M paclitaxel, there is a preven- hypothesized that Bcl-2 might be under tight regulation by tion of Bcl-2 phosphorylation, and the cells do not progress a phosphatase.135

to the G2/M transition. In addition, certain retinoids will also Treatment of prostate cancer cells that express exogenous block this phosphorylation as they induce a cell cycle block Bcl-2 with paclitaxel induces Bcl-2 phosphorylation and

at the G2/M transition. Human CLL cells that overexpress Bcl- apoptosis. In contrast, treatment of the prostate cancer cells, 2, show increased survival and are blocked at G0/G1. Treat- that do not express Bcl-2, with paclitaxel does not result in ment of non-cycling CLL cells with paclitaxel or taxotere apoptosis. It is possible to block Bcl-2 phosphorylation by could not result in Bcl-2 phosphorylation. In addition, in the making various site-directed mutants at Bcl-2 residues phos- presence of the protein synthesis inhibitor cycloheximide, phorylated by treatment with paclitaxel and other chemother-

Leukemia Review JA McCubrey et al 17

Figure 3 Interactions between serine/threonine kinases and apoptotic components–potential sites of interaction by chemotherapeutic drugs. The sites of interaction of the anti-cancer microtubule destabilizing drugs and other potential inhibitors are shown in yellow jagged circles. Some potential inhibitors include the purine analogue ﬂudarabine, the tyrosine kinase inhibitor genistein, the PKC/serine/threonine kinase inhibitor staurosporine, Raf inhibitors, the MEK inhibitor PD98059 and the phospholipase C inhibitor D609. Also the sites of interaction of the immunosuppressive drugs CsA and FK506 are shown. Moreover, certain antibodies such as antibodies to Fas may inhibit the effects of apoptosis inducing agents. Potential stimulators of some of the enzymes are shown in red. These include bryostatin, a PKC activator and aurintricarboxylic acid, an ERK2 activator. A red ‘P’ in a white circle indicates phosphorylated serine/threonine residues on proteins. A black ‘P’ in a white circle indicates phosphorylated tyrosine residues. apeutic drugs. Dr Haldar presented evidence that there may The effects of Bcl-2 on drug resistance were discussed by be additional kinases other than ERK2 which phosphorylate Dr Rakesh Srivastava (National Institute on Aging, NIH, Balti- Bcl-2 in response to paclitaxel on serines 70 and 87.136,137 more, MD, USA). Paclitaxel and other drugs, which disturb These kinases may phosphorylate Bcl-2 in response to pacli- microtubule dynamics, kill cells in a Fas/FasL dependent man- taxel on sites, which are different from the conventional ERK2 ner.140,141 Antibodies to FasL inhibit paclitaxel-induced recognition sequence. ERK2 was shown to phosphorylate both apoptosis. Bcl-2 overexpression leads to the prevention of the wild-type and mutated Bcl-2 proteins, indicating that ERK2 chemotherapy (paclitaxel)-induced production of FasL and may not be the kinase responsible for these paclitaxel-induced blocks paclitaxel-induced apoptosis in spite of paclitaxel- Bcl-2 phosphorylations. Indeed protein kinase A (PKA) was induced Bcl-2 phosphorylation.140,141 In addition, Bcl-2 over- found to be able to phosphorylate Bcl-2 in vitro and to be expression inhibits paclitaxel-induced apoptosis by blocking activated by taxotere in vivo in association with Bcl-2 phos- the nuclear translocation of nuclear factor of activated T cells phorylation.140 Instead, Dr Haldar presented evidence that (NF-AT) by binding and sequestering calcineurin, a critical there may be some involvement of the SAPK pathway in pacli- phosphatase necessary to dephophorylate NF-AT allowing taxel-induced phosphorylation and that this phosphorylation NF-AT to migrate into the nucleus (Figure 2). The BH4 domain of Bcl-2 requires the Bcl-2 homology region-4 (BH4) domain. of Bcl-2 is required for the interaction with calcineurin. In the The experiments employing SEK-dominant negative cell lines absence of NF-AT in the nucleus, the FasL gene is not tran- are in progress in Dr Haldar’s laboratory. Alternatively, one scribed and apoptosis does not occur. Thus paclitaxel and cannot exclude the possibility of the involvement of a 46 kDa other drugs that disturb microtubule function may kill cells in kinase with sequence similarity to SAPK in paclitaxel-induced part through the induction of FasL. Once Bcl-2 is phosphoryl- Bcl-2 phosphorylation. ated, it can not bind calcineurin.

Leukemia Review JA McCubrey et al 18 Dr Srivastava has also shown that paxitaxel and vincristine 2 Ihle JN. The Janus protein tyrosine kinase family and its role in induced increased expression of the pro-apoptotic Bax protein cytokine signaling. Adv Immunol 1995; 60: 1–35. in MCF-7 and MDA-MB-23 breast cancer cells. Over- 3 Larner AC, David M, Feldman GM, Igarashi K, Hackett RH, Webb DS, Sweitzer SM, Petricoin EF III, Finbloom DS. Tyrosine phos- expression of Bcl-2 in these cells counteracted the effects of phorylation of DNA binding proteins by multiple cytokines. low doses of these drugs. Paclitaxel and vincristine-induced Science 1993; 261: 1730–1733. activation of cyclic AMP-dependent PKA induced Bcl-2 hyp- 4 Larner AC, Finbloom DS. Protein tyrosine phosphorylation as a erphosphorylation and apoptosis. Inhibition of PKA activity mechanism which regulates cytokine activation of early response blocked these events. Dr Srivastava’s results suggested that genes. Biochim Biophys Acta 1995; 1266: 278–287. PKA activation in response to microtubule damage is an 5 Finbloom DS, Larner AC. Induction of early response genes by interferons, interleukins, and growth factors by the tyrosine phos- important event in Bcl-2 hyperphosphorylation. In contrast, phorylation of latent transcription factors. Implications for chronic DNA damaging drugs such as methotrexate and doxorubicin inﬂammatory diseases. Arthr Rheum 1995; 38: 877–889. had no effects on Bcl-2 hyperphosphorylation but still induced 6 Finbloom DS, Larner AC. Regulation of the Jak/STAT signalling cell death. Thus Bcl-2 is not required for induction of pathway. Cell Signal 1995; 7: 739–745. apoptosis. Paclitaxel and vincristine induce activation of cas- 7 Kapur R, Everett ET, Uffmann J, McAndrews-Hill M, Cooper R, pase 3 and cleavage of poly ADP-ribose. Both of these events Ryder J, Vik T, Williams DA. Overexpression of human stem cell are downstream of Bcl-2 phosphorylation. Dr Srivastava’s factor impairs melanocyte, mast cell, and thymocyte development: a role for receptor tyrosine kinase-mediated mitogen activated pro- work has suggested that there may be agents that disrupt or tein kinase activation in cell differentiation. Blood 1997; 90: damage the cytoskeleton that are p53-independent and func- 3018–3026. tion through serine/threonine kinase activation. The role of 8 Kapur R, Majumdar M, Xiao X, McAndrews-Hill M, Schindler K, paclitaxel-induced Bcl-2 phosphorylation is not yet resolved. Williams DA. Signaling through the interaction of membrane- Certain Bcl-2 phosphorylation events stimulated by growth restricted stem cell factor and c-kit receptor tyrosine kinase: gen- factors (IL-3, NGF and Epo) and PKC agonists prevent etic evidence for a differential role in erythropoiesis. Blood 1998; 91: 879–889. apoptosis whereas other Bcl-2 phosphorylation events, and in 9 Duronio V, Scheid MP, Ettinger S. Downstream signalling events some cases on the same serine residues, are associated with regulated by phosphatidylinositol 3-kinase activity. Cell Signal apoptosis. Whether these phosphorylation events can alter the 1998; 10: 233–239. conformation and/or the subceullular distribution of the Bcl- 10 Craddock BL, Welham MJ. Interleukin-3 induces association of 2 protein remains to be determined. Furthermore, the roles of the protein-tyrosine phosphatase SHP2 and phosphatidylinositol 3-kinase with a 100-kDa tyrosine-phosphorylated protein in hem- phosphorylation of other Bcl-2 family members (Bcl-XL, A1, M1 and others) remain to be elucidated. atopoietic cells. J Biol Chem 1997; 272: 29281–29289. 11 Anderson SM, Burton EA, Koch BL. Phosphorylation of Cb1 following stimulation with interleukin-3 and its association with Grb2, Fyn and phosphatidylinositol 3-kinase. J Biol Chem 1997; Summary 272: 739–745. 12 Minshall C, Arkins S, Freund GG, Kelly KW. Requirement for pho- It is now clear that cytokines use multiple, overlapping path- sphatidylinositol-3-kinase to protect hemopoietic progenitors ways to stimulate and regulate cell growth and differentiation. against apoptosis depends upon the extracellular survival factor. This meeting summarized some of the roles that J Immunol 1996; 156: 939–947. 13 Kubota Y, Angelotti T, Niederfellner G, Herbst R, Ullrich A. Acti- serine/threonine kinases have in signal transduction and the vation of phosphatidlyinositol 3-kinase is necessary for differen- prevention of apoptosis. Clearly cytokine receptors use both tiation of FDC-P1 cells following stimulation of type III receptor tyrosine and serine/threonine kinases as well as other signal tyrosine kinases. Cell Growth Differ 1998; 9: 247–256. transducing molecules to activate downstream kinases, tran- 14 Vanhaesebroeck B, Leevers SJ, Panayotou G, Waterﬁeld MD. Pho- scription factors and anti-apoptotic molecules. As these com- sphoinositide 3-kinases: a conserved family of signal transducers. plex, interconnected pathways become unraveled, hopefully Trends Biochem Sci 1997; 22: 267–272. the ability to isolate chemotherapeutic drugs, which can dif- 15 Pullen N, Thomas G. The modular phosphorylation and activation of p70S6K. FEBS Lett 1997; 410: 78–82. ferentially kill tumorigenic as opposed to normal cells, will 16 Downward J. Mechanisms and consequences of activation of pro- be achieved. A diagram showing some of the sites of known tein kinase B/Akt. Curr Opin Cell Biol 1998; 10: 262–267. chemotherapeutic drugs is presented in Figure 3. Once the 17 Delcommenne M, Tan C, Gray V, Rue L, Woodgett J, Dedhar S. roles of serine/threonine kinases are elucidated, the next big Phosphoinositide-3-OH kinase-dependent regulation of glycogen step will be to determine how they are abnormally expressed synthase kinase 3 and protein kinase B/AKT by the integrin-linked in neoplasia and whether chemotherapeutic strategies can be kinase. Proc Natl Acad Sci USA 1998; 95: 11211–11216. 18 Scheid MP, Duronio V. Dissociation of cytokine-induced phos- developed to utilize these kinases as molecular targets. phorylation of Bad and activation of PKB/akt: involvement of MEK upstream of Bad phosphorylation. Proc Natl Acad Sci USA 1998; 95: 7439–7444. Acknowledgements 19 Zha J, Harada H, Yang E, Jockel J, Korsmeyer SJ. Serine phosphorylation of death agonist Bad in response to survival factor The meeting was organized by Dr Allan Mufson and occurred results in binding to 14-3-3 not Bcl-xL. Cell 1996; 87: 619–628. 30 and 31 March at the Georgetown Holiday Inn, Wash- 20 del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G. ington, DC, USA. JAM was supported in part by a grant from Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science 1997; 278: 687–689. the National Institute of Health (R01 CA51025). We sincerely 21 Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg thank Ms Catherine Spruill for the outstanding artwork. ME. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 1997; 91: 231–241. 22 Scheid MP, Duronio V. Phosphatidylinositol 3-OH kinase activity References is not required for activation of mitogen-activate protein kinase by cytokines. J Biol Chem 1996; 271: 18134–18139. 1 Darnell JE Jr, Kerr IM, Stark GR. Jak-STAT pathways and transcrip- 23 Scheid MP, Schubert KM, Duronio V. Regulation of Bad phos- tion activation in response to IFNs and other extracellular signaling phorylation and association with Bcl-xL by the MAPK/ERK kinase. proteins. Science 1994; 264: 1415–1421. J Biol Chem 1999; 274: 31108–31113.

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