Leukemia (2000) 14, 9–21 2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu MINI-REVIEW AND MEETING REPORT 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 phosphoryl- ation 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 phosphoryl- ation 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.