Translocation and Reversible Minireview Localization of Signaling Proteins: A Dynamic Future for Signal Transduction

Stanford, California 94305 cesses. In nearly all cases studied, a significant fraction of cytosolic, membrane-bound or transmembrane proteins diffuse relatively freely within the cytosol or An increasing amount of experimental evidence has acmembrane. Fluorescence recovery after photobleaching cumulated over the last years suggesting that most sig(FRAP) studies of plasma membrane receptors, as well nal transduction processes utilize colocalization of seas of membrane-bound and cytosolic signaling proteins, quentially acting signaling proteins for the selective have shown that the diffusion coefficients of proteins activation of downstream functions (for reviews, see are quite variable and can be up to 0.05 mm/s for transPawson and Scott, 1997; Tsunoda et al., 1997). In many membrane receptors, up to 0.5 mm/s for membranecases, the activation of signaling proteins by upstream bound proteins and higher than 10 mm/s for cytosolic activators or their activation of downstream effectors proteins (for example, Jans et al., 1990; Niv et al., 1999; has been shown to involve binding interactions with Arrio-Dupont et al., 2000). As one of the fastest diffusing adaptor complexes, cytoskeletal structures, and subcelexamples, the 27 kDa EGFP (enhanced GFP; Clontech, lular membranes or with the targets and activators themCA) has been measured to have a diffusion coefficient selves. These localization mechanisms can be highly between 30 and 80 mm/s (i.e., Arrio-Dupont et al., 2000). regulated and rapidly reversible, leading to a dynamic It is interesting to note that the size of proteins, even view of signal transduction that diverges from the hisconjugated with GFP, is not a major obstacle for diffutoric “hardwired signaling concept” where receptors sion since the diffusion coefficient is inversely proporand other signaling proteins stay largely in place and tional to the radius (or the third root of its mass). Instead, spatial signal transmission is made possible by the rapid specific and unspecific binding interactions in the cytodiffusion of second messengers. In contrast, the current sol are the main reason for lower than expected cyto“softwired signaling concept” is built on the idea that solic diffusion coefficients. The average distance (,s.) signaling proteins translocate and undergo reversible that a protein diffuses from its origin along a given axis binding interactions as key steps of the signal transmiscan be calculated as, ,s. < (4 · D · t /p) (see Endnote sion process. The questions are then raised, whether 1), with D as the diffusion coefficient and t as the time translocation is an active or passive process, how fast of diffusion. As an example, a cytosolic signaling protein it can be, and which type of mechanisms can regulate with a D of 10 mm/s diffuses on average 4 mm in a 1 s the dynamic organization of large numbers of signaling time period. In contrast, membrane-bound and transproteins in space and time. membrane proteins diffuse in the same period less than The current evidence for this dynamic model of signal 0.8 and 0.25 mm, respectively. It should be noted that transduction stems from immunofluorescence, cell fracthe diffusion distances do not scale linearly but that the tionation and related biochemical approaches which average diffusion distance for proteins increases with have allowed in many cases to obtain snapshots of the the square root of the time (twice as far in 4 s compared changes in the subcellular localization of signaling proto 1 s). Figure 1 shows calculated views of a typical random teins. For example, the recruitment of cytosolic SH2 path of a diffusing cytosolic, membrane-bound and transdomain containing proteins by phosphorylated tyrosine membrane signaling protein (during a 4 s period). Markresidues at the plasma membrane or the nuclear transloedly, cytosolic signaling proteins can rapidly diffuse cation of many cytosolic signaling proteins and tranacross the cell, while transmembrane and membrane scription factors has been extensively explored by such bound signaling proteins can be regionally localized for methods. time periods of seconds to even minutes. Until recently the translocation of signaling proteins Given these considerations, how can one interpret an could only be followed “live” in cells in selective cases observed subcellular change in the localization of a GFP where signaling proteins could be fluorescently labeled conjugated signaling protein? A rough analysis of curin vitro and microinjected into cells for experimentation rently reported translocation time courses suggests that (Harootunian et al., 1993; Gough and Taylor, 1993). The nearly all of them are consistent with a random diffusion discovery that GFP and its variants can be used as process and are likely triggered by the encounter of a expressed fluorescence tags for signaling proteins and diffusing signaling protein with a newly enabled localsignaling domains led to a new strategy to track the ized binding site. Over time, the diffusing signaling prospatio-temporal dynamics of signaling processes by antein will screen a large region of a cell or membrane for alyzing stimulus-induced changes in their subcellular the presence of potential binding partners, giving rise localization (translocation and colocalization analysis). to the observed local enrichment or translocation. Since