Megakaryocytic Differentiation Induced by Constitutive Activation of Mitogen-Activated Protein Kinase Kinase

Abstract

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the plateletspecific integrin ␣ IIb ␤ 3 , all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, ␣-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes.It is now well established that the mitogen-activated protein (MAP) kinase cascade is a key regulator of mammalian cell proliferation (44). This pathway includes the MAP kinases extracellular-signal-regulated kinase 1 (ERK1) and ERK2, which are phosphorylated and activated by MAP kinase kinase 1 (MKK1) and MKK2 (44). ERKs are able to phosphorylate and activate the MAP kinase-activated protein (MAPKAP) kinases, including pp90 rsk (47), MAPKAP kinase 2 (46), and 3pK (45). MKK1 and MKK2 can be phosphorylated and activated by any of three protein kinases, Raf-1, MEK kinase, and Mos, and thus represent convergence points for diverse signalling pathways triggered upon cell surface receptor activation. Downstream targets of the MAP kinase cascade include several transcription factors which may be regulated by direct phosphorylation by the ERKs or MAPKAP kinases (9,13,20,32,50,56). Thus, the mitogen-activated MAP kinase cascade is a key mechanism for the control of transcription by extracellular signals.Several observations underscore the essential role for the MAP kinase pathway in cell transformation and cell cycle regulation. Signalling components further upstream, including receptor tyrosine kinases, Src, Ras, Raf-1, and the G

The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic

“…In contrast to NGF treatment, epidermal growth factor (EGF) treatment of PC12 cells induced an acute phasic activation of p42 MAPkinase which returned to baseline within 30 min ( Figure 1B). These data are in agreement with data of several other laboratories [16,21,22,34,35]. Bailie et al reported that primary cultures of hepatocytes possess binding sites for NGF, and we investigated whether NGF had a similar capability to modulate the MAP kinase cascade in these cells [26].…”

“…Indeed, the role of signalling by the MAP kinase cascade in terms of either cellular proliferation or cellular differentiation is currently under intensive study and recent reports using other cell types have suggested a role for chronic MAP kinase activation in causing exit from the cell cycle followed by cellular differentiation [11][12][13][14][15][16].…”

Stimulation of urokinase-type plasminogen activator receptor expression by PMA requires JNK1-dependent and -independent signaling modules

“…Additionally, PMA caused a rapid increase in ERK1 activity which paralleled the elevation in JNK activity. The involvement of the c-Raf-1-ERK signaling cascade in the modulation of gene expression by this phorbol ester is well established (Jones et al, 1994;Masuelli and Cutler, 1996;Whalen et al, 1997;de Vries-Smits et al, 1992;Bogoyevitch et al, 1995;Cai et al, 1997;Sozeri et al, 1992;Berra et al, 1995). These observations, combined with our unpublished ®nding that anisomycin, a selective activator of the JNKs (Bogoyevitch et al, 1995;Cano et al, 1994), does not increase u-PAR display, makes it likely that the propagation of the PMA stimulus to the nuclear transcriptional machinery regulating u-PAR expression is dependent on the cooperation of both JNK1-and ERK-dependent signaling modules.…”

“…However, a prevailing view is that the ability of phorbol ester to alter inducible gene expression is mediated through the classical signaling module (cRaf-1-ERK) and several observations by other investigators have supported this view. Thus, PMA induces ERK activity in a number of systems (Jones et al, 1994;Masuelli and Cutler, 1996;Whalen et al, 1997;de Vries-Smits et al, 1992;Bogoyevitch et al, 1995;Cano et al, 1995) and the co-expression of kinase-defective ERK1/ERK2 expression constructs inhibits the PMA-dependent stimulation of AP-1-regulated gene expression (Frost et al, 1994). Further, Protein Kinase C, the receptor for PMA, binds to and activates c-Raf-1 leading to ERK activation (Cai et al, 1997;Marquardt et al, 1994;Sozeri et al, 1992;Berra et al, 1995;Sauma and Friedman, 1996).…”

Scaffold mediated regulation of MAPK signaling and cytoskeletal dynamics: A perspective

“…For example, in some systems the platelet-derived growth factor (PDGF) stimulates sustained ERK activity and results in S-phase entry or cell differentiation whereas epidermal growth factor (EGF) stimulates transient ERK activity that does not drive cells in to S-phase and does not promote cell differentiation [204][205][206][207]. Duration and strength dependent modulation of cell fate appear to operate in neuronal PC12 cells, NIH3T3 fibroblasts [208], macrophages [209] and lymphocytes [210,211]. It is becoming apparent that cell fates determined by different growth factors that activate the same MAPK components requires a spatiotemporal control of MAPK targeting, sequestration and activation.…”