Leukemia (1998) 12, 1843–1850 1998 Stockton Press All rights reserved 0887-6924/98 $12.00 http://www.stockton-press.co.uk/leu REVIEW The roles of signaling by the p42/p44 mitogen-activated protein (MAP) kinase pathway; a potential route to radio- and chemo-sensitization of tumor cells resulting in the induction of apoptosis and loss of clonogenicity P Dent1,2, WD Jarvis3, MJ Birrer4, PB Fisher5, RK Schmidt-Ullrich1 and S Grant2,3,6 Departments of 1Radiation Oncology, 3Medicine, 2Pharmacology and Toxicology, and 6Microbiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA; 4National Cancer Institute, Biomarkers Branch, Division of Clinical Sciences, Rockville, MD; 5Columbia University College of Physicians and Surgeons, Department of Pathology and Urology, New York, NY, USA During the last 10 years, multiple signal transduction pathways terized dual specificity (threonine/tyrosine) protein kinase.4–6 within cells have been discovered. These pathways have been The use of the nomenclature MAPKK/MKK has declined, and linked to the regulation of many diverse cellular events such as proliferation, senescence, differentiation and apoptosis. this enzyme is more frequently referred to as MEK (mitogen This review will focus upon the many roles of signaling by the activated/extracellular regulated kinase). Shortly after the dis- p42/p44 mitogen-activated protein (MAP) kinase pathway. covery of MEK, a second isoform of this enzyme was identified Recent evidence suggests that signaling by the MAP kinase (MEK1 and MEK2).7 MEK1/2 were also found to be regulated pathway can both enhance proliferation by increased by reversible phosphorylation, and within 6 months of the dis- expression of molecules such as cyclin D1, but also cause covery of MEK2, the protein kinase responsible for catalyzing growth arrest by increased expression of molecules such as Cip-1/MDA6/WAF1 MEK1/2 activation was discovered, the proto-oncogene the cyclin kinase inhibitor protein p21 . These dif- 8,9 ferential effects on growth have been correlated to the ampli- Raf-1. tude and duration of the MAP kinase activity signal. Further- Raf-1 is a member of a family of serine-threonine protein more several laboratories are reporting data suggesting that kinases termed Raf-1, B-Raf and A-Raf.10–12 Each protein con- inhibition of the MAP kinase pathway, as well as a family of sists of an NH2-terminal domain (termed CR1), a COOH-ter- upstream MAP kinase activators, the protein kinase C family, minal catalytic domain (termed CR3), and a central domain represent an important route to both radio- and chemo-sensitiz- ation of tumor cells. Herein, we describe the historical dis- that is heavily phosphorylated in vivo (termed CR2). All ‘Raf’ covery and characterization of the MAP kinase pathway. In family members can phsophorylate and activate MEK1/2, addition we describe potential mechanisms by which inhibition although the relative ability of each member to catalyze this of protein kinase C, the MAP kinase pathway, and potentially reaction varies (B-RAF Ͼ Raf-1 Ͼ A-RAF).13,14 Seminal studies of p21Cip-1/MDA6/WAF1 expression, may alter the sensitivities of by the laboratories of Wolfman and Wigler demonstrated that leukemic and carcinoma cells to cytotoxic insults, leading to the CR1 domain of Raf-1 could reversibly interact with the increased apoptosis and loss of clonogenicity. 15,16 Keywords: MAP kinase; SAP kinase; growth factor; proliferation; Ras proto-oncogene in the plasma membrane. The ability differentiation; apoptosis; cyclin kinase inhibitor protein of Raf-1 to associate with Ras was dependent upon the Ras molecule being in the GTP-bound state. Studies from the lab- oratories of Hancock, Marshall, and Sturgill demonstrated that Introduction: MAP kinase historical background the ability of Raf-1 to be activated depended upon Raf-1 trans- location to the plasma membrane.17–19 The regulation of Raf- ‘MAP kinase’ was first reported by Sturgill and Ray in 1986.1 1 activity appears to be very complex, with several mech- This protein kinase was originally described as a 42-kDa insu- anisms co-ordinately regulating activity when in the plasma lin-stimulated protein kinase activity whose tyrosine phos- membrane environment. McCormick et al have demonstrated that association of Raf-1 with Ras is sufficient for partial stimu- phorylation increased after insulin exposure, and that phos- 20 phorylated the cytoskeletal protein MAP-2 (hence ‘MAP’ lation of Raf-1 activity. More recently, the binding of 14- 2 3-3 proteins to phospho-serine residues in Raf-1 have been kinase). Contemporaneous studies by Boulton and Cobb 20a,20b identified an additional 44-kDa isoform of MAP kinase, which suggested to be a key factor in Raf-1 activation. they named ERK1 (extracellular signal regulated kinase). Since Additionally, Bell and colleagues suggested that lipid second messengers such as phosphatidic acid may be able to bind to many growth factors and mitogens could activate MAP kinase, 21 the acronym for this enzyme has subsequently been con- Raf-1 and play a role in the activation process. Other investi- sidered to denote mitogen-activated protein (MAP) kinase. In gators have suggested that another lipid second messenger, ceramide, may also be able to play a role in Raf-1 acti- the following years, additional studies demonstrated that the 22,23 24,25 p42/p44 MAP kinases regulated another protein kinase vation, although this is disputed. Data from the lab- activity (p90rsk),3 and that they were themselves regulated by oratories of Sturgill, Morrison, and Marshall have suggested a protein kinase activity originally designated MKK (MAP that protein serine/threonine and tyrosine phosphorylations 4,5 play a role increasing Raf-1 activity when in the plasma mem- kinase kinase). 26–28 MKK phosphorylates the MAP kinases on tyrosine and thre- brane environment. Several other studies have also sug- onine residues and became the first biochemically charac- gested that PKC (protein kinase C) isoforms can directly regu- late Raf-1 activity.29,30 Phorbol esters and the macro-cyclic lactone bryostatin 1 can activate PKC, and have been shown to activate Raf-1 and the MAP kinase cascade in many cell Correspondence: P Dent, Department of Radiation Oncology, 401 31,32 College Street, Medical College of Virginia, Virginia Commonwealth types. Since phorbol ester-mediated downregulation of University, Richmond, VA 23298-0058, USA PKC blocks the abilities of some agonists, eg platelet-derived Received 26 June 1998; accepted 20 August 1998 growth factor (PDGF), but not others, eg epidermal growth The many roles of MAP kinase signaling P Dent et al 1844 factor (EGF), to activate Raf-1/MAP kinase, it is presumed that An overview for the role of the MAP kinase pathway in PKC plays an agonsit-specific role in mediating activation of proliferation and differentiation signaling Raf-1 and the MAP kinase pathway.33–36 At the same time that Raf-1 was shown to associate with Initial observations suggested that signaling by the MAP kinase Ras, the laboratories of Schlessinger and Pawson demon- pathway was intimately involved in the abilities of growth fac- strated that growth factors, via their plasma membrane recep- tors to stimulate proliferation.45 Indeed, for several years the tors, stimulate GTP for GDP exchange in Ras using guanine predominantly accepted view of signaling through the MAP nucleotide exchange factors.37,38 Thus, over an interval of ෂ9 kinase cascade based on studies with established fibroblastoid years, a pathway was delineated from plasma membrane and transformed cell models was that its activation promotes growth factor receptors, through guanine nucleotide exchange proliferation, and the greater the activation, the greater the factors and the Ras proto-oncogene, to the Raf-1/MEK/MAP proliferative response. For example, in NIH 3T3 fibroblasts, kinase/p90rsk MAP kinase pathway (Figure 1). transformation with either the v-Ha-Ras oncogene or the v-Raf During this period of time, other studies had begun to link oncogene caused constitutive activation of the MAP kinase growth factor induced MAP kinase and p90rsk activations to cascade and increased proliferation.46,47 In NIH 3T3 cells, the ability of these mitogens to regulate transcription factor expression of a constitutively active form of MEK1 also caused activities within the nucleus.39,40 The relative ability of MAP constitutive activation of the MAP kinase cascade and kinase signaling to mediate increased activity of a variety of increased proliferation.48 The positive role of MAP kinase acti- transcription factors is still under intensive study, potentially vation in cell cycle progression may be linked to increased because it appears that many signaling pathways under the expression of cyclin molecules, eg cyclin D1.48a,48b In leu- control of mitogens, eg c-Jun NH2-terminal kinase and p38- kemic cells, similar observations have been made. For reactivating kinase pathways, can co-ordinately regulate tran- example, several studies have demonstrated that stem cell fac- scription factor activities and gene expression along with the tor (SCF) and/or granulocyte–macrophage colony-stimulating classical MAP kinase pathway in an agonist and cell-type factor (GM-CSF) can stimulate MAP kinase activity in myeloid specific manner.41–44 and lymphoid leukemia cells, leading to increased prolifer- ation.49,51 In agreement with increased MAP kinase activity correlating with increased transformation and proliferation, elevated basal levels of MAP kinase activity are frequently found in acute myelogenous leukemia cells.52 However, more defined studies examining the extent and duration of MAP kin- ase activation, are now beginning to show that a simplistic view of increased activation of MAP kinase equating with increased proliferation is not necessarily valid. One example of such a discrepancy can be found in the case of PC12 pheochromocytoma cells, in which the role of MAP kinase signaling appears to conflict with the conven- tional view linking increased activity to enhanced prolifer- ation.