JCB: In Memoriam

From RAS to RHO: The making of the great cell biologist Alan Hall (1952–2015)

Chris Marshall

Division of Cancer Biology, Institute of Cancer Research, London SW3 6JB, England, UK

The fields of and cancer played a key role in the cloning of research are deeply saddened by the -interferon in Weismann’s laboratory. untimely death of Alan Hall at the I joined ICR about six months before young age of 62. There will be many Alan and embarked on a project to identify obituaries, so in this remembrance I human oncogenes using genomic DNA would like to describe some events in transfection into NIH3T3 cells, a meth- Alan’s career, and give a personal view odology pioneered by Bob Weinberg at on what I think made him an outstand- MIT. Alan and I met soon after he joined ing scientist and led to him having such ICR and discussed what we were plan- a major impact on our understanding ning to do. A couple of days later, Alan ll

of cell biology. came to see me and said he really wasn’t ha rs

Alan and I worked together for 12 interested in what Robin had proposed Ma years. We became close colleagues and as a project; he was much more interested ris

a deep friendship developed. Robin Weiss in what I was trying to do and suggested y of Ch es recruited both of us to the Institute of we work together. This suggestion was t ur o

Cancer Research (ICR) in London after probably one of the most significant events c he became director in 1980. Robin was in both our careers. Our skill sets and, as Photo very keen to introduce modern methods others observed, our rather different person- Alan Hall of cancer research into ICR and was on alities complemented each other beauti- the lookout to recruit promising molecu- fully. From the outset I think we both sensed of RAS proteins suggested that they lar biologists and cell biologists. Robin that we could work together effectively. were regulatory GTP proteins that might also brought Hugh Paterson with him Although we had our methodolo- be involved in signal transduction. As from his laboratory at the Imperial Can- gies firmly established, the search for we were interested in the biochemical cer Research Fund—Hugh and his cell novel transforming activities from human proteins of NRAS proteins and wanted biology and microscopy expertise were tumor DNA was frustrating. I presented to use recombinant proteins as a probe to play a central role in Alan’s work. the results of screening about 60 tumor for biological function, Alan and our stu- Alan started at ICR early in 1981. He DNAs at a laboratory meeting, and Robin dent Robin Brown produced cDNAs en- had gained an undergraduate degree in was so critical that Alan and I decided coding normal NRAS (Gly12) and an THE JOURNAL OF CELL BIOLOGY chemistry at Oxford University and then a we needed to have a meeting to discuss oncogenic mutant NRAS-Asp12 derived PhD with Jeremy Knowles in the enzymol- what to do. He suggested I bring my fam- from an acute myeloid leukemia NRAS ogy of -lactamase at Oxford and Harvard ily out to his home on the next Sunday, oncogene that Alan had isolated in col- University, from which he published his and so we took our children to the park laboration with Christoph Moroni. We first paper (Hall and Knowles, in the drizzling rain while we talked. In then worked with Frank McCormick to 1976). He then made a decisive change, the end, we decided we had a robust assay express NRAS proteins in Escherichia switching to working with recombinant and would look at another 20 tumor DNAs. coli. An interesting finding from stud- DNA technology by becoming a postdoc If nothing came out, we would have to ies with the protein was that microin- in the laboratories of Ken Murray (Uni- think about alternatives for our careers. jecting oncogenic RAS protein rapidly versity of Edinburgh) and then Charles Fortunately, in those 20 DNAs we iden- stimulated cell motility, an observation Weissmann (). Murray tified novel transforming activities, and and Weismann were major influences we were able to clone the third member of © 2015 Marshall This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No on the development and exploitation the RAS family NRAS (Hall et al., 1983). Mirror Sites license for the first six months after the publi- cation date (see http://www.rupress.org/terms). After six of recombinant DNA technology, and Alan The next thing to do was to study months it is available under a Creative Commons License how RAS proteins actually worked. The (Attribution–Noncommercial–Share Alike 3.0 Unported Correspondence to Chris Marshall: Chris.Marshall@ license, as described at http://creativecommons.org/licenses/ icr.ac.uk known GDP and GTP binding activities by-nc-sa/3.0/).

The Rockefeller University Press J. Cell Biol. Vol. 209 No. 4 481–483 www.jcb.org/cgi/doi/10.1083/jcb.201505049 JCB 481 that got us thinking about and in vitro and introduced into cells by mi- Alan was at the forefront of this cell motility (Trahey et al., 1987). Frank croinjection. Because activated mutant revolution, providing new insights into used the NRAS proteins to great effect versions of RHO did not occur naturally, the role of RHO family proteins in cyto- to identify RAS GTPase-activating pro- he made the glycine-to-valine mutant at skeleton regulation, phagocytosis, cell tein (GAP), a protein that increased the codon 14 equivalent to Val12 in RAS. polarity, and cell motility. He identified rate of GTP hydrolysis by normal RAS One of the first fruits of producing re- GAP proteins and effectors of RHO fam- but did not stimulate oncogenic RAS. combinant RHO protein was the demon- ily signaling. He did not just lead through These early studies on cell motility and stration with Klaus Aktories that the C3 his experimental work; his many reviews GAP piqued Alan’s interest, and were to toxin of Clostridium botulinum ADP- and commentaries also provided critical reemerge when he started to study the ribosylated Rho and that ribosylation of evaluations of the field and highlighted RAS-related RHO protein family. Rho accounted for the cytoskeletal ef- the key questions. To study signaling by RAS, we had fects of C3 (Aktories et al., 1989). These So what made Alan so great? I be- three principles. These principles were studies with C3 provided a powerful tool lieve there were several factors. First, very much influenced by the rigor of for studying RHO and provided an in- he was highly intelligent. Second he had Alan’s thinking, perhaps derived from his sight into the function of RHO. The next training in different disciplines: chemis- training in enzymology. First, we wanted key step was RHO protein microinjec- try, enzymology, and molecular biology. to use recombinant proteins to study early tion experiments performed by Hugh This, I think, made him very adaptable events stimulated by active GTP-bound Paterson that further demonstrated that and open to new ideas. This adaptabil- RAS. We believed that this would be RHO was acting on the actin cytoskeleton ity was reinforced by his expectation to more informative than comparing RAS- (Paterson et al., 1990). However, the move from laboratory to laboratory. He transformed cells with their nontrans- crucial steps were taken when Anne started his PhD in Oxford, then moved formed counterparts, which seemed to be Ridley joined Alan’s laboratory as a to Harvard, and went on to postdocs in a rather indirect and potentially messy ap- postdoctoral fellow. Edinburgh and Zurich before coming to proach. To study early events, we needed Anne took on two projects, one ICR London. He then moved across the a way of introducing recombinant RAS to study RHO itself and the other a re- city to University College London, fol- into cells. Here, Hugh Paterson was to cently identified closely related protein lowed by translocation to New York and prove invaluable because he had become RAC. Her work beautifully exempli- the Memorial Sloan Kettering Cancer adept at cell microinjection and could even fied the principles that Alan was using Center. Perhaps surprisingly to some microinject fragile quiescent Swiss 3T3 to analyze signaling: the responses to reading this, Alan had no formal training cells, at that time one of the best sub- introducing active proteins were rapid, in cell biology. In the early days of our strates for studying cell signaling. Later they occurred in the absence of growth collaboration, he was quite perplexed by on, Alan’s laboratory used the tech- factors, and when RHO or RAC were what he saw as the imprecise nature of nique of “scrape-loading” recombinant inhibited, the ability of external stimuli cell biology, and if you had said to me RAS proteins into cells that allowed us to evoke the responses, such as LPA at that time he would become Editor-in- to carry out biochemical assays (Morris stimulation of actin stress fiber forma- Chief of JCB, I would have been quite et al., 1989). Second, because it was thought tion, was blocked. Strikingly, although amazed. However the incisive nature of that oncogenic RAS would activate intra- they are quite similar proteins, RHO and his intellect allowed him to him iden- cellular signaling in the absence of growth RAC elicited very different responses tify important questions, and his adapt- factors, active RAS proteins should be on the actin cytoskeleton: RHO was ability meant he was prepared to do the able to do this in growth factor–free con- involved in forming stress fibers and work to take them on. Coupled with his ditions. Third, blocking normal RAS sig- focal adhesions whereas RAC drove intellect was great warmth of spirit and naling with dominant-negative mutants membrane ruffling. These studies, pub- generosity, perhaps derived from his should stop a RAS-dependent signaling lished in back-to-back papers in Cell Yorkshire background, where people response to growth factors. These prin- in 1992 (Ridley and Hall, 1992; Ridley are known not only for their directness ciples were crucial to dissecting RAS et al., 1992), revolutionized the study but also their friendliness. These as- signaling, and Alan then applied them of actin in cellular responses by link- pects of his character made him a great to great effect to study RHO family GT- ing activation of signaling pathways collaborator and a wonderful mentor of Pase signaling. of RHO family proteins to specific dy- young scientists. He was always willing In the mid-1980s, RHO was identi- namic actin responses. This work truly to discuss data and ideas with students fied as a protein highly related to RAS. revolutionized the field of cell biology and postdocs. Alan was devoted to JCB, Since the function of RHO was unclear, and spawned a new field investigating and one of his last pieces of work for Alan saw this as great opportunity to how RHO family GTPases transduce the journal was a wonderfully crafted study something new and decided to use stimuli into cellular responses. This piece with Kenneth Yamada discussing similar approaches for RHO that were field has had a major impact on our un- reproducibility in cell biology studies proving to be powerful tools to study RAS. derstanding of cell migration, cancer, (Yamada and Hall, 2015). His passing He produced recombinant RHO pro- microbial infection, developmental bi- has saddened many but his science and teins that could be studied biochemically ology, and neurobiology. the memories live on.

482 JCB • volume 209 • number 4 • 2015 References Aktories, K., U. Braun, S. Rösener, I. Just, and A. Hall. 1989. The rho gene product expressed in E. coli is a substrate of botulinum ADP- ribosyltransferase C3. Biochem. Biophys. Res. Commun. 158:209–213. http://dx.doi .org/10.1016/S0006-291X(89)80199-8 Hall, A., and J.R. Knowles. 1976. Directed selec- tive pressure on a -lactamase to analyse molecular changes involved in development of enzyme function. Nature. 264:803–804. http://dx.doi.org/10.1038/264803a0 Hall, A., C.J. Marshall, N.K. Spurr, and R.A. Weiss. 1983. Identification of transforming gene in two human sarcoma cell lines as a new member of the ras gene family located on chromosome 1. Nature. 303:396–400. http://dx.doi.org/10.1038/303396a0 Morris, J.D., B. Price, A.C. Lloyd, A.J. Self, C.J. Marshall, and A. Hall. 1989. Scrape-loading Swiss 3T3 cells with p21 ras induces rapid activation of protein kinase C. Oncogene. 4:27–31. Paterson, H.F., A.J. Self, M.D. Garrett, I. Just, K. Aktories, and A. Hall. 1990. Micro­ injection of recombinant p21rho induces rapid changes in cell morphology. J. Cell Biol. 111:1001–1007. http://dx.doi.org/10 .1083/jcb.111.3.1001 Ridley, A.J., and A. Hall. 1992. The small GTP-bind- ing protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 70:389–399. http:// dx.doi.org/10.1016/0092-8674(92)90163-7 Ridley, A.J., H.F. Paterson, C.L. Johnston, D. Diekmann, and A. Hall. 1992. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 70: 401–410. http://dx.doi.org/10.1016/0092-8674 (92)90164-8 Trahey, M., R.J. Milley, G.E. Cole, M. Innis, H. Paterson, C.J. Marshall, A. Hall, and F. McCormick. 1987. Biochemical and bio- logical properties of the human N-ras p21 protein. Mol. Cell. Biol. 7:541–544. Yamada, K.M., and A. Hall. 2015. Reproducibility and cell biology. J. Cell Biol. 209:191–193. http://dx.doi.org/10.1083/jcb.201503036

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