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Articles - Research University of Connecticut Health Center Research

8-2012

Fast rebinding increases dwell time of Srchomology 2 (SH2)-containing proteins near theplasma membraneDongmyung OhUniversity of Connecticut School of Medicine and Dentistry

Mari Ogiue-IkedaUniversity of Connecticut School of Medicine and Dentistry

Joshua A. JadwinUniversity of Connecticut School of Medicine and Dentistry

Kazuya MachidaUniversity of Connecticut School of Medicine and Dentistry

Bruce J. MayerUniversity of Connecticut School of Medicine and Dentistry

See next page for additional authors

Follow this and additional works at: http://digitalcommons.uconn.edu/uchcres_articles

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Recommended CitationOh, Dongmyung; Ogiue-Ikeda, Mari; Jadwin, Joshua A.; Machida, Kazuya; Mayer, Bruce J.; and Yu, Ji, "Fast rebinding increases dwelltime of Src homology 2 (SH2)-containing proteins near the plasma membrane" (2012). Articles - Research. 124.http://digitalcommons.uconn.edu/uchcres_articles/124

AuthorsDongmyung Oh, Mari Ogiue-Ikeda, Joshua A. Jadwin, Kazuya Machida, Bruce J. Mayer, and Ji Yu

This article is available at DigitalCommons@UConn: http://digitalcommons.uconn.edu/uchcres_articles/124

Fast rebinding increases dwell time of Src homology 2(SH2)-containing proteins near the plasma membraneDongmyung Oha, Mari Ogiue-Ikedab,c, Joshua A. Jadwinb,c, Kazuya Machidab,c, Bruce J. Mayerb,c,1, and Ji Yua,c,1

aCenter for Cell Analysis and Modeling, bRaymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, and cDepartment of Geneticsand Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030

Edited by John Kuriyan, University of California, Berkeley, CA, and approved July 10, 2012 (received for review February 27, 2012)

Receptor tyrosine kinases (RTKs) control a host of biologicalfunctions by phosphorylating tyrosine residues of intracellularproteins upon extracellular ligand binding. The phosphotyrosines(p-Tyr) then recruit a subset of ∼100 Src homology 2 (SH2) domain-containing proteins to the cell membrane. The in vivo kinetics ofthis process are not well understood. Here we use total internalreflection (TIR) microscopy and single-molecule imaging to moni-tor interactions between SH2 modules and p-Tyr sites near thecell membrane. We found that the dwell time of SH2 moduleswithin the TIR illumination field is significantly longer than predic-tions based on chemical dissociation rate constants, suggestingthat SH2 modules quickly rebind to nearby p-Tyr sites after disso-ciation. We also found that, consistent with the rebinding model,the effective diffusion constant is negatively correlated with therespective dwell time for different SH2 domains and the dwelltime is positively correlated with the local density of RTK phos-phorylation. These results suggest a mechanism whereby signaloutput can be regulated through the spatial organization of multi-ple binding sites, which will prompt reevaluation of many aspectsof RTK signaling, such as signaling specificity, mechanisms of spatialcontrol, and noise suppression.

reaction | epidermal growth factor receptor | diffusion-limited dissociation

The Src homology 2 (SH2) domain (1) controls many cellularactivities by binding specifically to tyrosine-phosphorylated

peptides (2). The tyrosine phosphorylation signal often originatesfrom the cell surface through activations of receptor tyrosinekinases (RTKs). Humans, for example, have 58 known RTKs (3),each of which could generate diverse phosphorylation patterns attheir cytosolic tails as well as at other associated proteins. Thesephosphorylation patterns are “read” mainly by a repertoire ofeffector proteins containing the SH2 domain (2, 4, 5), via re-cruitment of these effector molecules to the phosphotyrosine (p-Tyr) sites. For example, the epidermal growth factor receptor(EGFR), a well-known model system for studying phosphotyrosinesignaling, is activated by epidermal growth factor (EGF), a smallprotein ligand. The binding of EGF turns on the kinase activityof EGFR molecules, which then autophosphorylate themselvesat more than 20 tyrosine sites on the cytoplasmic tail. Thephosphotyrosines are thought to serve as docking sites for SH2-containing molecules, such as Grb2, which bind to these siteswith varying affinity. Downstream signals are propagated by theSH2 proteins, which in many cases are enzymes whose substratesare localized to the plasma membrane. In other cases such asGrb2, the SH2 protein serves to recruit additional downstreameffector proteins such as the Ras activator Sos. EGFR familymembers also play critical roles in cancer development andprogression and are frequently disregulated in tumors (6).During the last decade, an expanding assembly of quantitative

datasets has accumulated regarding tyrosine phosphorylation sig-naling, including the topological pattern of phosphorylation (7–10),the temporal dynamics of phosphorylation (10, 11), and charac-terization of the SH2/p-Tyr binding affinity (12, 13). On the otherhand, the in vivo assembly and turnover kinetics of the SH2complexes at p-Tyr sites remain poorly understood, partly be-cause existing methods measure mostly the ensemble propertiesof the system. To this end, measurements on the single-molecule

basis provide an advantage in understanding the dynamics ofthese heterogeneous interactions. With a total internal reflection(TIR) microscope, we can monitor the turnover of individual SH2molecules by tracking the molecule from its initial docking to a p-Tyr site at the cell’s plasma membrane to its dissociation from themembrane and leaving the TIR illumination field. The dwelltimes of single SH2 molecules within the TIR field thus can bemeasured and analyzed statistically. Here we report that ourexperimental results from EGF-stimulated cells can be quanti-tatively explained only when fast multiple rebinding of SH2molecules to p-Tyr sites is taken into consideration.

ResultsMeasuring Dwell Time of SH2 Molecules Near the Cell Membrane withTIR Illumination. We expressed in A431 cells the SH2 domain ofGrb2 (Grb2SH2), as a chimeric fusion to tdEos, a photoactivatablefluorescent protein. Single molecules were detected after a smallfraction of tdEos was photoactivated (14). Without extracellularligand stimulation, very few Grb2SH2 molecules could be seenunder TIR illumination (Fig. 1A and Fig. S1), but the number ofmolecules quickly increased upon the addition of EGF to the media(Fig. 1 A and B), presumably because the activation of EGFR led toan increased number of p-Tyr sites at the membrane. During thetime-lapse imaging (Movie S1), new molecules continuously appearat the plasma membrane, undergo diffusive motion for at mosta few seconds, and then disappear from the membrane. We at-tribute the appearance of a molecule to the initial binding re-action to a p-Tyr site and the disappearance to the escape of themolecule from the TIR illumination field or to photobleaching.Histograms of the dwell times, defined as the time delay be-

tween a molecule’s appearance and its disappearance, are shownin Fig. 1C. As expected the dwell time of Grb2SH2 falls betweenthat of a nonbinding mutant control (mtGrb2SH2), which weconsider to be due to nonspecific binding, and that of myr-tdEos(tdEos with a myristoylation signal peptide sequence from Srcthat directs constitutive membrane association), which is due tophotobleaching. However, because of the inevitable contributionfrom photobleaching, the dwell-time representation is not a di-rect measurement of the turnover kinetics. To overcome thisproblem, we calculated the hazard function (15), defined as

λðtÞ= pðtÞZ ∞

tpðuÞdu

; [1]

where p(t) is the probability distribution of the dwell times, es-timated by its histogram. The hazard function is additive for

Author contributions: B.J.M. and J.Y. designed research; D.O., M.O.-I., J.A.J., and K.M.performed research; D.O. analyzed data; and B.J.M. and J.Y. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1To whom correspondence may be addressed. E-mail: jyu@uchc.edu or bmayer@neuron.uchc.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1203397109/-/DCSupplemental.

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independent processes, and thus the contribution of photo-bleaching can be simply subtracted (SI Theory). The physicalmeaning of λ(t) is the average escape rate for molecules thatsurvived for at least time t. The value of λ(t) becomes a constant(independent of t) if the underlying kinetic process is a simplefirst-order process, in which case the hazard function is equal tothe first-order rate constant.After subtracting the contribution from photobleaching (Fig.

S2), we found that the hazard function for Grb2SH2 appears tobe a constant (Fig. 1D) at ∼0.52 s−1. This result was surprising intwo regards: First, the Grb2SH2 domain binds to multiple EGFRp-Tyr sites in vitro, with varying affinities (12), which presumablyarise partly from different dissociation rates. Heterogeneity isknown to affect the shape of the hazard functions (16). Fordissociation reactions, heterogeneity results in a decaying hazardfunction because a molecule that survived for a longer time ismore likely to be one bound to a high-affinity site and thus wouldhave a lower average dissociation rate. Second, the measuredaverage hazard function value, 0.52 s−1, is ∼20 times slower thanthe corresponding dissociation rate constant for Grb2SH2 pre-viously reported from in vitro studies (17, 18). In light of thisevidence, we suspected that the SH2 molecules might rebindefficiently, which can happen if there is a competition betweendiffusion and a high on rate between the SH2 and the p-Tyr (19,20). In such a scenario (Fig. 1E), the Grb2SH2 molecule rebindsmultiple times before eventually escaping from the TIR field.

The interaction of SH2 with multiple p-Tyr sites thereforeeliminates heterogeneity by averaging affinities over many sites.Indeed, on the basis of a reaction-diffusion model (SI Theory),we can theoretically predict a constant hazard function underthis condition (Fig. S3). Furthermore, with multiple rebindingevents, the apparent “dissociation rate” measured by the hazardfunction is much slower than the prediction based solely on thechemical off rate. In fact, we found (SI Theory and Fig. S4) thatthe hazard function should be dependent not only on the offrate, kd, but also on the on rate, ka, as well as on the diffusionconstant, D, of the molecule when hopping between sites,

λðtÞ ≈ kd=ð1+akan=DÞ; [2]

where n is the p-Tyr density and a is the imaging depth of theTIR field. On the basis of this model, on average ∼20 rebindingevents were detected in our experiment for each Grb2SH2 mol-ecule before it escaped. Furthermore, the equation predicts thatfactors that increase the binding rate, konn, will further decreasethe ability of molecules to escape the TIR field; on the otherhand, fast diffusion of the dissociated molecules, which is con-trolled by D, promotes their escape. The importance of rebindingcan also be evaluated outside the context of the TIR experiment.Lagerholm and Thompson (21) proposed using a unitless param-eter, b ≡ kan=

ffiffiffiffiffiffiffiffiffikdD

p, to evaluate the significance of rebinding:

For systems with b << 1, rebinding can be safely ignored and the

I. Simple dissociation dwell-time = 1/koff

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Fig. 1. Rebinding of the Grb2SH2 domain to phosphotyrosine sites increase its dwell time at the cell membrane. (A) Recruitment of Grb2SH2 (Left) andthe nonbinding mutant control, mtGrb2SH2 (Right). Epi-fluorescence image shows the expressed molecules distributed mostly in the cytosol (Top row). TIRillumination of unstimulated cells shows little recruitment of Grb2SH2 to the cell membrane (Middle row). Upon EGF stimulation, a large number of mobileGrb2SH2 molecules, but not mtGrb2SH2, can be detected with TIR fluorescence (Bottom row). (Scale bar: 2 μm.) (B) The time course of Grb2SH2 recruitment afterEGF stimulation. The amount of the Grb2SH2 recruitment was measured by integrated fluorescence intensity under TIR illumination (lines) or by counting thenumber of single molecules (bars). The different traces represent measurements from four different cells. (C) Survival-time distribution for Grb2SH2,mtGrb2SH2, and Myr-tdEos in A431 cells stimulated for 20 min with EGF. (D) Hazard function (lines) of Grb2SH2 computed from survival-time distribution (bars).Solid lines are the simple moving average. (E) Schematic diagram illustrating the rebinding mechanism.

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dissociation kinetics are dictated by the chemical rate constantkd, whereas when b >> 1 the kinetics of the dissociation processare strongly influenced by rebinding. On the basis of the hazardfunction, we estimated the value of b for Grb2SH2 to be ∼44.6,assuming a kd value of 10 s−1 (17) and a D value of 15 μm2/s,corresponding to the cytosolic diffusion rate of a small protein (22).

SH2 Mobility Is Correlated with Its Apparent Membrane Dwell Time.To verify that the rebinding dynamics are indeed important to theinteractions between SH2 domains and p-Tyr sites, we measuredthe hazard functions, λ(t), as well as the effective mobility, Deff, offive different SH2 domains from four SH2 proteins that arethought to directly interact with EGFR (Movie S2 and Fig. 2).We hypothesized that, if SH2 domains dissociate from the mem-brane without rebinding, the mobility should solely be determinedby the diffusion constant of the EGFR receptor protein; thus, thevariation in mobility of different SH2 domains should be small. Onthe other hand, if the SH2 domains rebind frequently, the mobilityis determined not only by the diffusion constant of the receptors,but also by the frequency and the duration of the “hopping”motionbetween p-Tyr binding sites. We found that Deff varied by morethan an order of magnitude among different SH2 domains (Fig.2A), supporting a rebinding mechanism. Furthermore, all five SH2domains we studied exhibited constant hazard functions, albeit atdifferent average values (Fig. 2B), again consistent with a rebindingmechanism. Finally, the average hazard rates and the mobilitieswere correlated (Fig. 2C), as was predicted by the reaction-diffu-sion model (SI Theory):

Deff =D0λ−1 +a2=2D0

λ−1 +a2=2D: [3]

Here D0 is the diffusion constant of EGFR, which is ∼0.03 μm2/s(23), and D is the diffusion constant of the unbound SH2, which is∼15 μm2/s (22). Using these numbers, and assuming TIR illumi-nation depth a ∼ 0.5 μm, we can directly calculated the value ofDeff as a function of λ. We found that the calculated values fromthis equation agree well with our experimental data (Fig. 2C).

Spatial Distribution and Clustering of Target Phosphotyrosine BindingSites. The prominence of SH2 rebinding suggests a high densityof potential p-Tyr binding sites (∼103 sites/μm2 based on Eq. 2).We asked whether this density is the average density of the wholecell or a local density elevated due to microscopic domains of thereceptors clustering (24). To answer this question, we quantified

the effects of the duration of EGF stimulation on the hazardfunction values and the effective mobilities of Grb2SH2. Indeed,as the stimulation duration increased, the dwell time of Grb2SH2molecules increased as well (Fig. 3A and Fig. S5), presumablydue to an increase in p-Tyr density. The number of Grb2SH2molecules recruited to the membrane typically reached a plateauat around 5–10 min of stimulation (Fig. 1B), suggesting that thenumber of p-Tyr sites reaches a maximum value at that time.Results from other assays, such as far-Western analysis (Fig. S6)and mass spectrometry (11), led to the same conclusion. In-terestingly, however, the SH2 dwell time continued to increaseand stabilized only after >20 min (Fig. 3A), suggesting that therebinding of SH2 molecules was indeed responding to changesin local density of the binding sites. Even though the number andthus the average density of p-Tyr sites no longer increased after10 min, our data suggest that the local density continued to increase,most likely by clustering of the EGFR receptors. As expected ifthe local density of binding sites were increasing, the mobility ofthe Grb2SH2 molecule decreased correspondingly over time,presumably because they spend less time in cytosol beforerebinding (Fig. 3B). The changes in the mobility and the changesin the dwell time are negatively correlated with each other (Fig.3C). These results highlight the importance of the spatial orga-nization (local density) of the receptors, as opposed to their totalexpression, in regulating the membrane dwell time of SH2molecules. Supporting this idea, we found that in cells (H226)with much lower EGFR expression than A431, the hazardfunction and the dwell time of Grb2SH2 are almost the same as inA431 cells (Fig. S7).To understand the spatial distribution of the phosphotyrosine

binding sites in cells, we recorded the first observed locations ofeach individual SH2 molecule detected at the cell membraneand plotted their coordinates (Fig. 3 D–I). We found that whenthe cell was stimulated for a short period, no apparent spatialpattern could be discerned from the plot (Fig. 3E). For cells thatwere stimulated for a longer time (e.g., >20 min), however, wefound spatial regions to which the SH2 molecules were re-peatedly recruited with a higher-than-average frequency, in-dicating clusters of p-Tyr sites on activated EGFR (Fig. 3 F–H).No clustering was observed for mtGrb2SH2 (Fig. 3I). This resultfurther lends support to the argument that the decrease in haz-ard function values is correlated with clustering of the receptors.

Rebinding Dynamics of Multivalent SH2 Molecules. Next, we in-vestigated the turnover dynamics of multivalent SH2 molecules.Most signaling proteins are multivalent: They have multiple

A B C

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Fig. 2. Correlation between the effective mobility and the apparent dissociation rate. (A) Effective diffusion constant (Deff) for different SH2 modules.The ensemble-averaged MSD-Δt curves were computed from single-molecule traces for five different SH2 modules (squares). The effective mobilitieswere obtained by computing the slope (straight lines) from the first to the fourth time lags in the MSD-Δt curves, corresponding to the ∼200-ms timescale.The results illustrated the significant differences of Deff among different SH2 modules. (B) Hazard functions of different SH2 modules. The hazard functionswere computed from survival-time distributions as in Fig. 1. (C) Correlation between Deff and the average dwell time, calculated as the inverse of the averagehazard function values (1/λ). The solid line is the theoretical prediction from Eq. 3, assuming D = 15 μm2/s, D0 = 0.03 μm2/s, and a = 0.5 μm. The theoreticalvalues agree well with the experimental data.

14026 | www.pnas.org/cgi/doi/10.1073/pnas.1203397109 Oh et al.

modular binding domains that allow interaction with more thanone molecule at the same time. Some SH2 proteins, such as Shp2or PLCγ1, have two SH2 domains and can thus be recruited tothe membrane through either monovalent or bivalent bindingto p-Tyr (Fig. 4A). We measured the hazard function and theeffective mobility of the Shp2SH2(NC) molecule (Movie S3),which has both the N- and the C-terminal SH2 domains of theShp2 phosphatase. In contrast to the monovalent Shp2SH2(N) orShp2SH2(C), the hazard function of Shp2SH2(NC) is no longera constant (Fig. 4B), presumably due to a heterogeneous mixtureof both monovalent and bivalent complexes. The monovalentcomplex was formed when an SH2 molecule was initially re-cruited to the membrane, and the bivalent complex was formedlater. Thus, we attribute the decay in the hazard function to thegradual process of bivalent complex formation. The decay time

constant is on the order of a few hundred milliseconds, indicatingthat the formation of the second connection is relatively slowcompared with the formation of the first. The difference is prob-ably because once the SH2 molecule is tethered to the mem-brane through the first p-Tyr-SH2 interaction, its diffusion ratedecreases significantly, which results in a reduced reaction ratefor the formation of the second binding.We expect the monovalent complex to rebind via diffusion in

cytosol, whereas the bivalent complex rebinds as a membrane-associated complex (Fig. 4A). Therefore, the rebinding modelwould predict that the molecules with longer survival time shouldhave lower mobility than molecules with shorter survival time.Indeed with MSD-Δt analysis we found the mobility of moleculeswith long survival times (>5 s) is approximately eight to ninetimes less than that of molecules with short survival times (<1 s)

A B C

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EGF Induction time (min) EGF Induction time (min) 1/ (sec)

Fig. 3. Temporal evolution of Grb2SH2 turnover dynamics. (A) Average λ-values decrease over time. The hazard function was measured every 1.5 min afteraddition of EGF. The average λ-values between 0.1 s and 2.0 s were calculated. The decreasing values indicate that the apparent dissociation rate is lower forcells stimulated for a longer time. (B) Effective mobilities (Deff) decrease over time. The effective mobilities were measured as in Fig. 2. (C) The decrease ineffective mobility and the increase in survival time are correlated. (D) Average fluorescence intensity of Grb2SH2 molecules at the cell’s plasma membrane byTIR microscopy. The image shows the overall spatial distribution of the Grb2SH2 molecules. (E–H) Spatial distribution of the binding sites during various timedelays after EGF stimulation. The spatial location of each SH2 binding event was recorded and plotted as a black dot at the spatial location. The plotted regioncorresponds to the box in D. The same number of molecules was plotted for E–H. (I) Same as in E–H except the cell was expressing mtGrb2SH2. The cell wasstimulated for 40 min. (Scale bars: 5 μm.)

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(Fig. 4C and Fig. S8). Such a bifurcation in mobility was notobserved for monomeric SH2 molecules (Fig. 4C and Fig. S8).Furthermore, the mobility of Shp2SH2(NC) molecules with shortsurvival times (<1 s) is roughly the same as that of their mono-meric counterpart [Shp2SH2(N)]. These results are consistent withthe rebinding model. The hazard functions of two other bivalentSH2 molecules [PLCγ1SH2(NC), which has the two naturally oc-curring SH2 domains of PLCγ1, and an engineered tandem Grb2construct, Grb2SH2-SH2] exhibited similar decaying values (Fig. S9),consistent with the above model.Finally, we also measured the hazard function of full-length

Grb2, in which the SH2 domain is flanked by SH3 domains that

bind to proline-rich peptides in effectors such as Sos (Fig. 5). Wefound that the hazard function of full-length Grb2 exhibits verylittle decay, suggesting that the recruitment of Grb2 to the cellmembrane depends predominately on the SH2 domain. That is,SH3-mediated interactions do not appear to be sufficient to tetherGrb2 to the membrane. Consistent with this interpretation, wealso found no significant difference in the recruitment kineticsbetween full-length Grb2 and the Grb2SH2 domain (Fig. 5A), anda mutation in the SH2 domain alone (mtGrb2) was sufficient toprevent recruitment of full-length Grb2 to the cell membrane(Fig. 5A). We found that the average dwell time of full-lengthGrb2 was longer than that of Grb2SH2, consistent with the pre-vious finding that full-length Grb2 and Grb2/Sos complexes bindtighter to p-Tyr (17, 25). The flattened hazard function furtherindicates that the heterogeneities of multiple p-Tyr sites wereaveraged out, presumably due to multiple rebinding, indicatingthat the results we obtained from isolated SH2 domains can beextrapolated to full-length SH2 proteins.

DiscussionIn this study, we measured the apparent dwell time of SH2domains within a TIR illumination field near the cell membrane.We found the average dwell time is longer than predicted on thebasis of the chemical dissociation rate, and for monomeric SH2domains the dissociation kinetics exhibited little heterogeneity.Although other possibilities cannot be completely excluded, thesimplest and most likely explanation is that the interaction be-tween SH2 domains and p-Tyr sites involves repeated rebinding.We propose that the rebinding is the result of competition be-tween the very fast intrinsic chemical kinetic rate of the associationreaction and the relatively slow diffusion rate of the biomoleculesin vivo. Part of the reason that the chemical association rate is highis that there is an abundance of available p-Tyr binding sites,because each receptor molecule has multiple phosphorylationsites and because of the clustering of the receptors that furtherincreases the local concentration of binding sites. Because theSH2 molecules do not directly dissociate from the receptors in ourmodel, the traditional concept of turnover time has an ambiguousmeaning in this case. There are, in fact, two different turnovertimes. The SH2 turnover time on an individual p-Tyr site is quiteshort, because the molecule quickly dissociates. The fast dissoci-ation is consistent with the fact that p-Tyr sites can be very quicklydephosphorylated (26) and thus are not protected by the SH2binding for a very long time. On the other hand, the SH2 turnovertime with respect to the receptor cluster is relatively long, becausethe SH2 molecule “hops” between the binding sites. Theorypredicts that clustering stabilizes the binding between p-Tyr sitesand the SH2 molecules, which we verified in experiments.The finding that the SH2 protein hops between clustered

binding sites along the cell membrane suggests that a single SH2molecule, when recruited to the membrane, could potentiallyinteract with multiple p-Tyr sites that are near each other. Thiskind of mechanism may also play a role in other intracellular

(DS

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Fig. 4. Rebinding of multivalent SH2 module Shp2SH2(NC). (A) two differentschemes of rebinding for bivalent SH2 molecules, depending on whether itforms a monovalent complex (Upper) or a bivalent complex (Lower). (B) Thehazard functions for the Shp2SH2(NC) module in an A431 cell stimulated byEGF (20 min), in comparison with its monomeric counterparts. The noncon-stant λ-values for Shp2SH2(NC) arise from the mixed population of mono-valent complexes and bivalent complexes. The decay reflects the temporalprocess of bivalent complex formation. (C) MSD-Δt analysis of Shp2SH2(NC)(Top), Shp2SH2(N) (Middle), and Shp2SH2(C) (Bottom). For each molecule, theMSD curves were calculated for the subpopulations with long (blue), in-termediate (red), and short (black) survival times. Only Shp2SH2(NC) exhibiteddifferent MSD curves depending on survival times.

(Full length)

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Norm

alize

d de

nsity

(a.u

.) Fig. 5. Turnover kinetics of full-length Grb2molecules. (A) Comparison of the recruitment ki-netics of full-length Grb2, full-length mtGrb2, andGrb2SH2 in A431 cells. Data were collected fromA431 cells as in Fig. 1B. The amount of the recruitmentwas measured by integrated fluorescence intensityunder TIR illumination after cells were treated with25 ng/mL EGF. (B) Hazard functions of full-lengthGrb2 and Grb2SH2 in A431 cells. Similar to that ofGrb2SH2, the hazard function of full-length Grb2 isa constant. The lower average value for full-lengthGrb2 signifies a longer average dwell time, consistentwith a more stable binding to p-Tyr sites.

14028 | www.pnas.org/cgi/doi/10.1073/pnas.1203397109 Oh et al.

protein interactions. For example, studies of Cdc4 interactionwith Sic1 revealed that Cdc4 can interact with multiple phos-phorylated sites on Sic1 even though Cdc4 contains only oneinteraction site (27). Together with our findings, it seems plau-sible that dynamic equilibrium through multiple rebinding eventsis a common theme of phosphoprotein interactions. The rebindingmechanism lessens the importance of single high-affinity bindingsites. In terms of RTK signaling, the SH2 dwell times depend onthe average affinities of many phosphorylated sites; lack of a singlehigh-affinity site will have only a moderate impact on the averageduration of binding. Therefore, the rebinding mechanism mayexplain why mutation of RTK phosphosites predicted to bindspecifically to particular SH2 domains had relatively little effecton downstream signaling. It was only when all sites were elimi-nated and individual phosphosites added back that specific effectson downstream signaling could be observed (28, 29).Finally, these results underscore the importance of receptor

clustering, as kinetic stabilization of the SH2/p-Tyr interactiondepends on high local density of phosphorylated sites. Receptorclustering may further affect the downstream outcome of thesignaling process (30). For example, it was proposed (31) that inT cells, prolonged ligand/receptor association suppresses “noise”from adventitious receptor activation (a kinetic proofreadingmechanism). In a similar manner, the increased SH2 proteindwell time near the cell membrane, coupled to high in vivodephosphorylation rates (26), may serve to dampen spuriousnoise associated with nonspecific binding of SH2 proteins tomembrane molecules. Finally, because the recruited SH2 pro-teins do not associate with just one p-Tyr site, but instead “glide”over many p-Tyr sites, the chemical interaction of such an SH2protein with downstream substrate or effector molecules at thecell membrane may not follow simple chemical kinetic laws. The

details of such interactions should be an interesting subject offuture research.

Experimental ProceduresConstructs and Cell Culture. SH2 domain DNA sequences were obtained fromthe Human SH2 collection (Open Biosystems) and subcloned to the C terminusof tdEos coding sequence to form the chimeric fusion constructs. The myr-tdEos clone contains the myristoylation signal sequence from the N terminusof Src. A431 and H226 cells were maintained in DMEM culture medium andreplated on freshly cleaned glass-bottom dishes (Matek) at medium to lowdensity (<50% confluent) right before microscopy experiments.

Microscopy and Image Analysis. All imaging experiments were carried out ona custom-made total-internal-reflection fluorescence microscope, equippedwith a 532-nm laser (Crystal Lasers) for fluorescence excitation and a 405-nmlaser (Coherent) for photoactivation. Fluorescent spots corresponding tosingle molecules were detected via intensity thresholding. The positions ofthe molecules were tracked over time, using the nearest-neighbor method.The scan area used during tracking when searching for the position of themolecule in the next time frame was chosen on the basis of estimateddiffusion rate of the molecule so that the molecule has a >99.5% chance tobe within the scan area. The spatial density of the fluorescence spots waskept low (0.05–0.1/μm2) to avoid mistracking. To estimate the effectivemobility, ensemble averaged MSD was calculated as a function of time lags(Δt), which was subsequently used to determine the effective diffusionconstant.

More details of the experimental procedure are included in the SIExperimental Procedures.

ACKNOWLEDGMENTS. This research is supported by National Cancer In-stitute Grant U01CA154966 (to B.J.M. and J.Y.) and National Institutes ofHealth Grants R01CA82258 and RC1CA146843 (to B.J.M.) and R01GM085301(to J.Y.).

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