A Raf-1 Mutant That Dissociates MEK/Extracellular Signal-Regulated Kinase Activation from Malignant Transformation and Differentiation but Not Proliferation

Abstract

It is widely thought that the biological outcomes of Raf-1 activation are solely attributable to the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. However, an increasing number of reports suggest that some Raf-1 functions are independent of this pathway. In this report we show that mutation of the amino-terminal 14-3-3 binding site of Raf-1 uncouples its ability to activate the MEK/ERK pathway from the induction of cell transformation and differentiation. In NIH 3T3 fibroblasts and COS-1 cells, mutation of serine 259 resulted in Raf-1 proteins which activated the MEK/ERK pathway as efficiently as v-Raf. However, in contrast to v-Raf, RafS259 mutants failed to transform. They induced morphological alterations and slightly accelerated proliferation in NIH 3T3 fibroblasts but were not tumorigenic in mice and behaved like wild-type Raf-1 in transformation assays measuring loss of contact inhibition or anchorage-independent growth. Curiously, the RafS259 mutants inhibited focus induction by an activated MEK allele, suggesting that they can hyperactivate negative-feedback pathways. In primary cultures of postmitotic chicken neuroretina cells, RafS259A was able to sustain proliferation to a level comparable to that sustained by the membrane-targeted transforming Raf-1 protein, RafCAAX. In contrast, RafS259A was only a poor inducer of neurite formation in PC12 cells in comparison to RafCAAX. Thus, RafS259 mutants genetically separate MEK/ERK activation from the ability of Raf-1 to induce transformation and differentiation. The results further suggest that RafS259 mutants inhibit signaling pathways required to promote these biological processes.

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

“…As we have shown previously (Brummer et al, 2003), expression of B-Raf wt , but not of B-Raf K483W , resulted in the differentiation of 10-20% of transfectants ( Figure 2). In line with its high signalling activity (Figure 1b), approximately 50% of PC12 transfectants expressing B-Raf V600E displayed prominent neurites, a percentage that has been also observed upon expression of other Raf oncogenes (MacNicol et al, 2000;Dhillon et al, 2003) or the constitutively active enhanced green fluorescent protein (EGFP)-DB-Raf protein (Figure 2; Brummer et al 2003). In comparison with B-Raf wt transfectants, those expressing B-Raf R188L displayed a 3-4-fold reduction in neurite outgrowth, indicating that B-Raf must interact with Ras-GTPases to exhibit its full biological activity (Figure 2b).…”

“…Regulation of B-Raf signalling T Brummer et al and Guan, 2000;Dhillon et al, 2003). As we have shown previously (Brummer et al, 2003), expression of B-Raf wt , but not of B-Raf K483W , resulted in the differentiation of 10-20% of transfectants ( Figure 2).…”

Regulation of phorbol ester-mediated TRAF1 induction in human colon cancer cells through a PKC/RAF/ERK/NF-κB-dependent pathway

“…MEK1/2 is an important downstream effector of cRaf (Dhillon et al, 2003). Therefore, to explore the possibility that the c-Raf/MEK/MAPK pathway contributes to PKC-mediated TRAF1 induction, we pretreated colon cancer cells with the MEK inhibitors PD98059 or U0126 prior to PMA treatment.…”

Faecal tumour M2 pyruvate kinase: a new, sensitive screening tool for colorectal cancer

“…The tetramer : dimer ratio of M2-PK is under the control of several oncoproteins, such as pp60 vÀsrc kinase, HPV-16 E7 and A-Raf ( Figure 1B) (Eigenbrodt et al, 1998b;Zwerschke et al, 1999;Le Mellay et al, 2002). Interestingly, pp60 cÀsrc kinase and A-Raf are consistently altered in gastrointestinal tumours (Bolen et al, 1987;Iravani et al, 1998;Irby et al, 1999;Luckett et al, 2000;Dehm et al, 2001;Dhillon et al, 2003).…”