Cells need to sense and respond to their environment. To do this, they have dedicated proteins that interpret outside signals and convert them into appropriate responses that are only active at a specific time and location within the cell.

However, in many diseases, including cancer, these signaling proteins are switched on for too long or are active in the wrong place. To better understand why this is the case, researchers manipulate proteins to identify the processes they regulate. One way to do this is to engineer proteins so that they can be controlled by light, turning them either on or off.

Ideally, a light-controlled tool can activate proteins at defined times, control proteins in specific locations within the cell and regulate any protein of interest. However, current methods do not combine all of these requirements in one tool, and scientists often have to use different methods, depending on the topic they are researching.

Now, Shaaya et al. set out to develop a single tool that combines all required features. The researchers engineered a light-sensitive ‘switch’ that allowed them to activate a specific protein by illuminating it with blue light and to deactivate it by turning the light off. Unlike other methods, the new tool uses a light-sensitive switch that works like a clamp. In the dark, the clamp is open, which ‘stretches’ and distorts the protein, rendering it inactive. In light, however, the clamp closes and the structure of the protein and its activity are restored. Moreover, it can activate proteins multiple times, control proteins in specific locations within the cell and it can be applied to a variety of proteins.

This specific design makes it possible to combine multiple features in one tool that will both simplify and broaden its use to investigate specific proteins and signaling pathways in a broad range of diseases.

Dissection of biological processes is greatly aided by optogenetic methods that allow researchers to mimic and manipulate the function of individual proteins. Multiple broadly applicable approaches have been developed to enable tight regulation of protein localization and protein interactions (Guntas et al., 2015; Wang et al., 2016; Strickland et al., 2012; Kawano et al., 2015); however, the direct control of enzymatic activity remains a challenge. Only a few existing strategies achieve direct regulation of enzymatic activity, but they either lack the ability to activate enzymes locally within a cell, or they are difficult to apply to multiple enzyme classes (Wang et al., 2019; Dagliyan et al., 2016; Zhou et al., 2017; Diaz et al., 2017; Wu et al., 2009; Hongdusit et al., 2020), thus limiting their use for addressing mechanistic questions in cell biology. This highlights the need for an optogenetic approach which is broadly applicable, easy to implement and provides all the advantages of optogenetics in one tool including tight temporal and local subcellular regulation. An attractive strategy that can be harnessed for this purpose is the application of a rationally designed light-sensitive domain that can allosterically control protein activity. This protein engineering approach offers several important advantages. It enables targeted regulation of one domain within a multidomain protein (Karginov et al., 2010). Regulation is achieved ‘remotely’ without steric interference with the catalytic pocket of the enzyme and substrate binding. Also, allosteric switch domains are genetically encoded into the targeted protein simplifying the application of the tool. Only four optogenetic methods for allosteric regulation of activity have been reported so far and they suffer from critical limitations (Dagliyan et al., 2016; Wu et al., 2009; Hongdusit et al., 2020; Winkler et al., 2015; Reynolds et al., 2011). Two of these strategies achieve subcellular control of certain targeted proteins but they cannot be applied to many other enzymes due to their design (Wu et al., 2009; Hongdusit et al., 2020). One approach is broadly applicable but it does not achieve local control and triggers inactivation rather than activation of the protein (Dagliyan et al., 2016). A similar approach described by Reynolds et al achieves regulation in both directions for dihydrofolate reductase (DHFR) and PDZ domain but efficiency of this approach for applications in mammalian cells and its ability to regulate proteins at subcellular level has not been demonstrated (Reynolds et al., 2011). Furthermore, existing allosteric switches lack the ability to tune the activation/inactivation kinetics, an important feature required for mimicking different temporal signaling modes of enzymes.

Among the various potential targets of value for cell biology applications, protein kinases constitute an important family of enzymes that regulate key physiological functions and thus their activity is tightly controlled. Aberrant kinase regulation is the underpinning of many diseases, including the development and progression of malignant tumors (Lee and Yaffe, 2016). Many kinases are therefore important therapeutic targets and significant research effort is directed towards uncovering their function in cells. Such functional studies are challenging because a single kinase often induces drastically different responses depending on the location, level and timing of its activation (Marshall, 1995; Rauch et al., 2016; Cohen-Saidon et al., 2009; Bugaj et al., 2018; Toettcher et al., 2013). Furthermore, transient, sustained, or oscillatory kinase activation can result in distinct outcomes. Therefore, interrogating kinase-mediated signaling requires approaches capable of mimicking complex spatiotemporal control of kinase activity down to the subcellular resolution. Traditional methods using genetic manipulation and small molecule inhibitors lack this level of regulation. Optogenetics, on the other hand, overcomes these limitations by leveraging the precision of light-mediated activation (Toettcher et al., 2011). Some existing optogenetic tools regulate the dimerization or localization of kinases (Toettcher et al., 2013; Wend et al., 2014; Grusch et al., 2014; Chang et al., 2014; Kim et al., 2014). These approaches control the catalytic activity only indirectly and can only be applied to a narrow group of kinases. Other approaches employ light-sensitive caging or allosteric switches for the regulation of kinase catalytic domains (Wang et al., 2019; Dagliyan et al., 2016; Zhou et al., 2017). However, these methods do not achieve subcellular control of activity and some of these approaches are also limited in their broader applicability due to structural requirements (Wang et al., 2019; Dagliyan et al., 2016; Zhou et al., 2017). Thus, the development of a broadly applicable approach to enable optogenetic regulation of protein kinases remains challenging and highly desirable.

Here, we report the development of a protein engineering approach that enables light-mediated allosteric control of enzymatic activity and combines critical advantages of optogenetics in one tool: it provides tight temporal regulation of activity with tunable kinetics, enables local control that can be achieved with subcellular resolution, and allows for broad applicability to different types of enzymes. We achieve this by employing an engineered light sensitive switch domain that is genetically encoded and enables the allosteric regulation of kinase catalytic domains. It provides fast, specific, and tunable control of kinase activity in living cells using low intensity blue light. Furthermore, this method enables reversible and repetitive activation of a kinase and provides local control of kinase activity at a subcellular level. Application of this tool allowed us to uncover temporal dynamics of phosphorylation-mediated signaling following short term (10 s) as well as prolonged activation of a kinase. Importantly, we show that this approach can be applied not only to different kinases but also to other enzymes.

Our study describes an optogenetic approach that provides several advantages for the interrogation of signaling pathways and demonstrates its broad applicability to address important biological questions. The key features of this method include: (1) allosteric regulation of the enzymatic activity, (2) tight temporal control of activity with tunable kinetics, (3) local regulation of activity at a subcellular level, and (4) broad applicability to different enzymes. Importantly, unlike other approaches, LightR combines all these advantages in one tool, thus, simplifying application of optogenetics in biological research.

We achieved direct regulation of enzymatic activity via an allosteric control by inserting the LightR switch into small loops within the protein structure. This modular design provides significant flexibility in the selection of the insertion site and allows for specific regulation of catalytic activity without compromising key functions of the protein such as its interactions with binding partners or its native localization in the cell (Karginov et al., 2010). Furthermore, variation of the flexible linkers, both between the kinase and VVD as well as between the two VVDs, or changing the insertion site for LightR in the targeted enzyme could result in reversing or altering the regulatory mechanism of LightR, as was previously demonstrated for insertion of LOV2 domain in DHFR (Reynolds et al., 2011). While we implemented only one linker type and only one insertion site in LightR-Src, this is a venue for future work to generate more modular tools. Application of LightR approach to different classes of enzymes suggests its broad applicability for the regulation of a wide variety of protein functions in living cells. Thus, unlike other light-regulated allosteric switches (Wu et al., 2009; Hongdusit et al., 2020), this method combines multiple important advantages of optogenetic regulation with its broad applicability to a wide range of enzymes.

We showed that regulation by LightR domain is tunable, enabling different modes of regulation. LightR switch with slow off kinetics will be useful for long-term activation of LightR-enzymes; since brief periodic pulses of light will be sufficient to maintain activity while avoiding phototoxicity caused by long exposure to blue light. The FastLightR switch, on the other hand, is more suitable for studies that mimic transient, oscillatory or localized activation of a protein. It could also be used to study the kinetics of negative regulators of signaling pathways immediately after a signaling input is turned off. Activation of a LightR-enzyme requires low intensity light (Figure 2—figure supplement 1B, 0.6 mW/cm²), minimizing the phototoxic effects of blue light illumination. This level is lower than the intensity used in other optogenetic studies using light-sensitive switches to regulate enzymes (Dagliyan et al., 2016; Zhou et al., 2017; Wang et al., 2012). The activation of LightR is limited to the blue light spectrum, thus enabling its multiplexing with other red-shifted optogenetic tools or FRET biosensors.

Several optogenetic approaches for the regulation of protein kinases have been described previously; however, all had specific shortcomings. Several methods regulate the localizationof a kinase rather than its catalytic activity (Kawano et al., 2015; Graziano et al., 2017; Mühlhäuser et al., 2019; Moitrier et al., 2019; Katsura et al., 2015; Zhang et al., 2014). The method described by Zhou et al provides efficient control of kinases by light-regulated steric hindrance but it does not enable regulation on a subcellular level (Zhou et al., 2017). Furthermore, one of the regulatory domains has to be inserted in the FG loop, a substrate-interacting region in several kinases including Src family kinases (Zhou et al., 2017; Shah et al., 2016). Thus, this approach may not be applicable to some critical kinases. The insertion site that we selected for LightR domain is intentionally positioned away from any substrate binding elements, which should minimize any steric hindrance by this domain. Dagliyan et al., achieved regulation of Src through insertion of LOV domain at the same site that we used for the LightR approach (Dagliyan et al., 2016). However, this method also did not achieve local regulation at a subcellular level. Furthermore, this approach only enabled inactivation of a kinase by illumination with light. To activate a kinase at a specific time, researches have to first inhibit the kinase by keeping cells under blue light for a significant period of time, then release the inhibition by switching the light off. This requirement increases the possibility of potential cytotoxic effect of light and makes the implementation of this tool problematic for studies where activation of a kinase is desired. While the insertion of LOV domain at a different site may result in light-induced activation, as was previously demonstrated for DHFR (Reynolds et al., 2011), this has not been achieved for protein kinases at the moment. Thus, combined advantages of LightR enable broader application of this tool for interrogation of kinase signaling.

Tight and efficient control of LightR allowed us to mimic fast signaling dynamics of Src kinase in cells, and to establish stimulation of different signaling patterns over time. Several identified targets link early effects of Src activation to focal adhesion regulation and membrane protrusion formation. Our phosphoproteomics analysis revealed early Src-dependent phosphorylation of talin-1 at Y26 and Y70 residues (Figure 2D). These residues are located in the N-terminal F0 FERM domain of talin-1 (Bouaouina et al., 2008; Goult et al., 2010). When talin-1 is in the inactive closed conformation, the F0 domain remains exposed and is proposed to act as an early detector of changes in the environment (Bouaouina et al., 2008). Thus, phosphorylation of talin-1 on Y26 and Y70 by Src may represent an early step in the inside-out integrin activation mechanism performed by talin (Vinogradova et al., 2002; Vinogradova et al., 2004). Later phosphorylation (1–5 min) of proteins involved in formation of new focal adhesions and membrane protrusions (such as lamellipodin, p130Cas, palladin, tensin-3, cortactin, GIT1, and paxillin) correlates with the time at which we observed cell spreading and increased protrusion velocity (Figures 4A and 5H). Elevated phosphorylation of many proteins at 1 hr is indicative of oncogenic transformation processes mediated by prolonged activation of Src (Hunter and Sefton, 1980; Sefton et al., 1980). Our phosphoproteomics studies also revealed that continuous Src activity induces only transient activation of MAP kinases ERK1 and ERK2 (Figure 2—figure supplement 3). This suggests that short term Src activation induces activation of ERK1/2 whereas prolonged activity triggers inactivation of this pathway. Previous studies demonstrated that stimulation of cells with EGF results in transient activation of ERK1 and ERK2 (Olsen et al., 2006; Sasagawa et al., 2005). In the future studies, it will be interesting to study whether Src also plays a role in negative regulation of ERK1/2 downstream of EGF. Importantly, our approach allowed us to identify temporal categories of Src targets; distinguishing between targets that are phosphorylated at early, intermediate, or delayed time points. Such temporal compartmentalization can be elucidated by the optogenetic approach and provides unprecedented insights into the complexity and diversity of signaling networks.

Periodic activation of kinases occurs during different cellular processes (Kaimachnikov and Kholodenko, 2009; Roche et al., 1995; Hilioti et al., 2008; Jacquel et al., 2009; Kholodenko, 2000; Maeda et al., 2004). Depending on the kinase and the physiologic context, periodicity of these cycles ranges from minutes to hours, which often determines the functional outcome (Hilioti et al., 2008; Jacquel et al., 2009; Kholodenko, 2000; Maeda et al., 2004; Karginov et al., 2014). Using FastLightR, we demonstrated that we can modulate activation cycles of protein kinases, Src (Figures 3C and 4A,C; Video 1) and bRaf, (Figure 6D; Video 3) with light. Tight regulation of FastLightR allows us to control the periodicity and the amplitude of these activation pulses. Therefore, FastLightR can be potentially applied to any enzyme of interest to directly reconstruct its oscillatory signaling and identify its functional role.

The ability to regulate proteins on a subcellular level is one of the main capabilities desired in optogenetic tools. However, this has been challenging to achieve for methods employing steric hindrance and allosteric control as a mechanism of regulation (Dagliyan et al., 2016; Zhou et al., 2017). Efficiency and fast reversibility of FastLightR allowed us to control Src activity at a specific location in the cell and demonstrate that its activity is sufficient to induce local protrusion. One of the interesting observations is the fact that continuous local activation of Src induces waves of protrusions instead of causing persistent protrusions (Figure 5E,H). Several groups have previously described periodic protrusions at the cell edge (Tsai and Meyer, 2012; Giannone et al., 2004; Machacek and Danuser, 2006), and correlated this phenotype with periodic contractions due to phosphorylation of myosin light chain (MLC) (Tsai and Meyer, 2012; Giannone et al., 2004; Machacek and Danuser, 2006). Our results demonstrate that both kinases that phosphorylate MLC (ROCK and MLCK) participate in the formation of the recurrent waves of protrusion in response to local Src activation. However, ROCK appears to be less important for maintaining the overall elevated protrusion velocity downstream of Src activation (Figure 5—figure supplement 2B). Inhibition of MLCK resulted in much more noticeable reduction in average protrusion velocity (Figure 5—figure supplement 2B). Previous studies suggested that MLCK predominantly acts at the cell periphery where it mediates formation of stable protrusions and persistent cell migration (Totsukawa et al., 2004). Our data is consistent with these prior studies and suggest that MLCK is a predominant factor regulating cell protrusions downstream of Src.

Several previous studies described approaches for the regulation of Cre recombinase activity by light (Wu et al., 2020; Morikawa et al., 2020; Kawano et al., 2016; Taslimi et al., 2016; Sheets et al., 2020; Kennedy et al., 2010). However, these methods are designed based on the split protein reassembly strategy, which has its limitations. Split proteins tend to reassemble spontaneously, leading to undesirable basal activity of engineered split Cre (Dagliyan et al., 2018; Liu and Tucker, 2017). Moreover, when activated, not all split halves of the protein reassemble, which often leads to suboptimal activation levels of the engineered enzyme (Dagliyan et al., 2018). Furthermore, this method requires co-expression of two proteins and, to achieve optimal results, both split halves must be co-expressed in equimolar ratios in a cell. LightR overcomes some of these limitations, because LightR-Cre is, by design, a single construct that expresses a complete Cre enzyme fused with LightR domain. Although high levels of LightR-Cre expression sometimes lead to low basal activity (Figure 6—figure supplement 2), this could be solved by optimizing the expression level of LightR-Cre and/or by modifying the linkers connecting LightR domain to the Cre recombinase. We believe that such optimization is feasible because we did not detect any basal activity for the LightR-kinases regardless of their expression level in cells.

In summary, the LightR-approach to regulate enzyme activity demonstrates versatility and broad applicability to a variety of enzymes and can thus help unravel signaling pathways and networks as well as study defined biological processes that are direct consequences of the regulated enzymes.

The raw mass spectrometry data and associated tables have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier: PXD018162. All data generated or analyzed during this study are included in the manuscript and supporting files.

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     https://doi.org/10.1021/bi8007017

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Acceptance summary:

This paper presents an original and potent optogenetic switch that allows allosteric control of enzyme activity. Albeit the use of fungal-based LOV domains to orthogonally control different cellular processes is not new, this light-regulated allosteric switch presents unique features that makes of LightR a powerful tool to dissect and explore basic and fundamental biological questions. The dataset presents fine examples of how Src activity can be controlled with the switch of a light, allowing tunable, temporal and spatial control of its activity (with subcellular resolution), revealing important details about its dynamics. The modular features of LightR are clearly exemplified in its use not only to modulate Src activity (ON in the light/ OFF in the dark), but also in the control of the kinases Abl and bRaf, as well as of Cre recombinase. For sure LightR (and future variations of this type of optogenetic allosteric switch) will continue to enlighten not only aspects of kinase biology, but also unforeseen features of diverse cellular processes.

Decision letter after peer review:

Thank you for submitting your article "LightR: A Light-Regulated Allosteric Switch Enables Temporal and Subcellular Control of Enzyme Activity" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Luis F Larrondo as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Jonathan Cooper as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, when editors judge that a submitted work as a whole belongs in eLife but that some conclusions require a modest amount of additional new data, as they do with your paper, we are asking that the manuscript be revised to either limit claims to those supported by data in hand, or to explicitly state that the relevant conclusions require additional supporting data.

Our expectation is that the authors will eventually carry out the additional experiments and report on how they affect the relevant conclusions either in a preprint on bioRxiv or medRxiv, or if appropriate, as a Research Advance in eLife, either of which would be linked to the original paper.

Summary:

The work by Shaaya et al. describes a new strategy for engineering allosteric regulation of proteins by blue light, by genetically inserting a light-sensitive clamping domain (LightR) into a loop of an enzyme's surface. The authors present examples of this strategy on three different well-studied kinases (Src, Abl, and bRaf) and on Cre recombinase. LightR is based on a tandem VVD (LOV domain) fusion and works as a clamp-like switch that opens in the dark and closes (because of VVD-VVD intramolecular dimerization) in response to blue-light. This rationally designed light-sensitive domain, based on a LOV fungal domain, constitutes a nice addition to the repertoire of optogenetic switches described in past years and exemplifies an interesting strategy to achieve intramolecular optogenetic control.

The ability to orthogonally regulate by light any enzyme that can be inactivated by inserting the LightR domain into a surface loop is novel and welcome, and the LightR variant with faster off-kinetics is a useful addition. As shown by the authors, this allows achieving reversible and repeatable light activation at different time scale with subcellular resolution.

The data described by the authors indicate that this strategy could represent a broadly applicable approach.

Essential revisions:

The major concern relates to some parts of the manuscript that require clarification. There are aspects of how the data are discussed, and how the methods are described, that ought to be improved in order to help colleagues grasp key aspects of the work and value its significance and reach. It is also important to contextualize the results in order to understand the novelty and contributions of the presented strategy.

1) The MD simulation methods used are not sufficiently detailed. Moreover, the scale of the molecular dynamics work presented in Figure 1 does not contribute to the claims made by the authors. More specifically, it relies on single short simulations of a homology model constructed with little experimental knowledge of the actual orientation of the VVD domain relative to the kinase catalytic domain.

2) Some experimental detail on the expression of LightR constructs in cells is unclear or lacking. From the Materials and methods section, it appears that most of the work was done in LinXE cells transiently expressing LightR constructs and that the phosphoproteomics work was done in HeLa cells stably expressing the constructs, but this is unclear in the main text. Also, the specifics of the LightR-kinase and LightR-Cre recombinase constructs are unclear: the figures seem to indicate that LightR-Src and FastLightR-Src are myc-tagged, while LightR-Abl and bRaf are GFP-tagged, but this is not mentioned in the main text or the Materials and methods. The authors should briefly describe the major components of the constructs used when they first appear in the main text and must also provide the full sequences of the constructs in the supplement.

3) Little validation data is presented for either transient or stable expression of the constructs: only single-time-point western blots for transient LightR-kinase expression in Figures 2, 3 and 6, no data for transient LightR-Cre expression and no data for stable LightR-Src expression. This data likely already exists and should probably be presented in supplementary figures.

4) The experiments using ROCK and MLCK inhibitors are problematic and may not support the conclusion that ROCK is less important a mediator of Src's effect on cell-edge velocity than MLCK. Y-27632, the ROCK inhibitor used, is reported to have an IC50 of 140 nM, so the authors' choice of a 10-µM working concentration is fairly reasonable, but the MLCK inhibitor peptide is reported to have an IC50 of 50 nM and to be cell-permeable, so the authors' choice of a 100-µM working concentration is worryingly high and may have off-target effects that are actually mediating the observed greater decrease in cell-edge velocity. The authors need to justify the concentrations used or otherwise justify the relative importance of ROCK and MLCK for mediating Src's effect on membrane protrusion. This is a relatively minor portion of this paper, which focuses on the development of the LightR tool, and removing this claim would not significantly affect the strength of the paper.

5) The authors propose that the novelty of the method lies in a combination of features: 1) regulation is achieved allosterically (rather than a sterically or by localization/sequestration) 2) regulation is achieved with subcellular precision and 3) the regulation shows tunable kinetics. However, each of these properties have been established elsewhere and here the main contribution lies in the combination of all three. The tunable kinetics in this work made use of a previously characterized mutation and thus isn't particularly unique. Other work has shown precise spatiotemporal localization (e.g. Cavanaugh et al. (2020) Cell Protoc Cell Biol 86:e102), but by localization rather than allostery. And allosteric regulation of an enzyme by light is shown in both (Dagliyan et al., 2016, and Lee et al. (2008) Science 322:438), but as the authors point for Dagliyan et al. "it does not achieve local control". Thus, the novelty of the current work requires a *very* precise definition of what is new.

Likewise, the work is most similar to (Dagliyan et al., 2016). Both manuscripts describe the regulation of Src by a LOV domain, and the authors write that the main distinction between this work and their own is that Dagliyan et al. "does not achieve local control and triggers inactivation rather than activation of the protein". The directionality of the regulation (activating vs. deactivating) is not necessarily fundamental – others have observed both activation and deactivation by LOV2 insertion, even within the same protein (Reynolds et al., 2011). These aspects need to be explained/discussed.

6) Modulation of CRE by LightR is not mentioned in the Discussion. Moreover, there have been several reports where CRE activity (split strategy) has been utilized to control its activity (PMID: 32709899; PMID: 32358538; PMID: 27723747; PMID: 27065233, including work where CRE is regulated by VVD dimerization (e.g. Sheets et al., 2020 and Kennedy et al., 2010). Those examples should be referenced, and the advantage of having a light-activated CRE in only one construct, as opposed to two (split strategy) should be mentioned.

7) The work does not give a good sense of whether (or not) insertion of the tandem VVD domain perturbs kinase function. For example, the domain insertions in (Lee et al. (2008) Science 322:438) result in a dramatic decrease in the WT enzyme activity. The authors might address this by comparing their in vivo measurements to existing data for activated native Src or bRaf. Another method may be using in vitro kinase assays to directly measure activity in lit and dark states. If these experiments cannot be currently conducted, the authors should at least clearly discuss this point.

8) "This indicates that the open conformation of LightR clamp in the dark can cause changes in the catalytic domain of Src that will be reversed upon illumination with blue light". The authors should also discuss if this approach could be implemented (by changing the type of linker, or where LightR is inserted) such that the "distorted" conformation can be reached in the light state, whereas the "WT/active" one could be achieved in the dark.

The description of the LightR should briefly mention the size of the utilized VVD (compared to what has been used in optogenetic systems), and the type of linker that was added in between both VVDs. This info is extractable from the figures, but it would help if it is explicitly mentioned: for example, how many different GS linker sizes were simulated in silico, and how many were experimentally tried before defining the definitive LightR construct.

Essential revisions:

The major concern relates to some parts of the manuscript that require clarification. There are aspects of how the data are discussed, and how the methods are described, that ought to be improved in order to help colleagues grasp key aspects of the work and value its significance and reach. It is also important to contextualize the results in order to understand the novelty and contributions of the presented strategy.

1) The MD simulation methods used are not sufficiently detailed. Moreover, the scale of the molecular dynamics work presented in Figure 1 does not contribute to the claims made by the authors. More specifically, it relies on single short simulations of a homology model constructed with little experimental knowledge of the actual orientation of the VVD domain relative to the kinase catalytic domain.

We apologize for not providing enough details about the MD simulations. We included more details in the revised manuscript under the “Development of a Light-Regulated Kinase” subsection in the Results section, and under the “Molecular dynamics modeling” subsection in the Materials and methods section.

We had previously failed to mention that the data presented were the combined result of three independent 100 ns simulations. In Figure 1—figure supplement 1, we also included a plot of the RMSD of the system over the course of the simulation as validation of our homology model. Convergence of the RMSD of the system is indicative of a structure occupying an energetic minima and thus represents a reasonable structure for analysis. While we acknowledge that there are intrinsic limitations to homology modeling, it provides a method to guide protein engineering efforts and evaluate possible mechanisms of regulation before experimental data are obtained. Similar approaches have been used in previously published work to assess engineered molecules and propose potential mechanism of action

(https://pubmed.ncbi.nlm.nih.gov/27980211/, https://doi.org/10.1007/s10989-018-9791-9, https://doi.org/10.1007/s10989-006-9058-8, https://doi.org/10.3389/fmolb.2020.00004, https://pubmed.ncbi.nlm.nih.gov/26673131/).

2) Some experimental detail on the expression of LightR constructs in cells is unclear or lacking. From the methods section, it appears that most of the work was done in LinXE cells transiently expressing LightR constructs and that the phosphoproteomics work was done in HeLa cells stably expressing the constructs, but this is unclear in the main text. Also, the specifics of the LightR-kinase and LightR-Cre recombinase constructs are unclear: the figures seem to indicate that LightR-Src and FastLightR-Src are myc-tagged, while LightR-Abl and bRaf are GFP-tagged, but this is not mentioned in the main text or the Materials and methods. The authors should briefly describe the major components of the constructs used when they first appear in the main text and must also provide the full sequences of the constructs in the supplement.

We appreciate this comment. In the revised manuscript, we specified the cell lines used in every experiment in the main text and figure legends (Figures 2-6; Figure 2—figure supplements 1-4; Figure 3—figure supplement 1; Figure 5—figure supplement 1B; and Figure 6—figure supplements 1, 2). We also specified whether the constructs are transiently or stably expressed. Furthermore, we described the major components of the constructs when they first appear in the main text and provided their full DNA sequence at the end of the Materials and methods section.

3) Little validation data is presented for either transient or stable expression of the constructs: only single-time-point western blots for transient LightR-kinase expression in Figures 2, 3 and 6, no data for transient LightR-Cre expression and no data for stable LightR-Src expression. This data likely already exists and should probably be presented in supplementary figures.

We apologize for not providing validation data for the HeLa stable cell line (LightR-Src HeLa) we generated. We included a western blot in the revised manuscript in Figure 2—figure supplement 1C that shows comparable expression levels of LightR-Src and endogenous Src.

We also provided western blots showing transient expression levels of LightR-Src,

LightR-Abl, and LightR-bRaf at each time point. These can be found in Figure 2C;

Figure 3A-C; Figure 6B, C; Figure 2—figure supplement 1B; Figure 3—figure supplement 1; Figure 6—figure supplement 1.

To compare LightR-Cre-iRFP expression levels between different time points, we provided images of LightR-Cre-iRFP in Figure 6F and in Figure 6—figure supplement 2. In the previous submission, we failed to indicate that all images were taken using the same illumination and acquisition settings and were adjusted to the same brightness/contrast level; so the expression levels can be compared between samples. Even though LightR-Cre-iRFP was not measured by Western blot, fluorescence microscopy suggests that similar expression levels were achieved in different experiments. This is now clarified in the revised version.

4) The experiments using ROCK and MLCK inhibitors are problematic and may not support the conclusion that ROCK is less important a mediator of Src's effect on cell-edge velocity than MLCK. Y-27632, the ROCK inhibitor used, is reported to have an IC50 of 140 nM, so the authors' choice of a 10-µM working concentration is fairly reasonable, but the MLCK inhibitor peptide is reported to have an IC50 of 50 nM and to be cell-permeable, so the authors' choice of a 100-µM working concentration is worryingly high and may have off-target effects that are actually mediating the observed greater decrease in cell-edge velocity. The authors need to justify the concentrations used or otherwise justify the relative importance of ROCK and MLCK for mediating Src's effect on membrane protrusion. This is a relatively minor portion of this paper, which focuses on the development of the LightR tool, and removing this claim would not significantly affect the strength of the paper.

We understand the concern about the concentration we used. We decided to use 100 μm of the MLCK inhibitor peptide based on the published work referenced on the manufacturer’s website (https://faseb.onlinelibrary.wiley.com/doi/full/10.1096/fj.09-146118#fsb2fj09146118-fig-0005). In this work, authors pre-treated microglia with 100 µM MLCK inhibitor to achieve inactivation. Furthermore, a separate study demonstrated that MKCK inhibitor peptide 18 maintains high specificity for up to 4,000 fold of its IC50, which is a concentration of 200 μm (J. Med. Chem. 42(5), 910-919 (1999)).

5) The authors propose that the novelty of the method lies in a combination of features: 1) regulation is achieved allosterically (rather than a sterically or by localization/sequestration) 2) regulation is achieved with subcellular precision and 3) the regulation shows tunable kinetics. However, each of these properties have been established elsewhere and here the main contribution lies in the combination of all three. The tunable kinetics in this work made use of a previously characterized mutation and thus isn't particularly unique. Other work has shown precise spatiotemporal localization (e.g. Cavanaugh et al. (2020) Cell Protoc Cell Biol 86:e102), but by localization rather than allostery. And allosteric regulation of an enzyme by light is shown in both (Dagliyan et al., 2016, and Lee et al. (2008) Science 322:438), but as the authors point for Dagliyan et al. "it does not achieve local control". Thus, the novelty of the current work requires a *very* precise definition of what is new.

Likewise, the work is most similar to (Dagliyan et al., 2016). Both manuscripts describe the regulation of Src by a LOV domain, and the authors write that the main distinction between this work and their own is that Dagliyan et al. "does not achieve local control and triggers inactivation rather than activation of the protein". The directionality of the regulation (activating vs. deactivating) is not necessarily fundamental – others have observed both activation and deactivation by LOV2 insertion, even within the same protein (Reynolds et al., 2011). These aspects need to be explained/discussed.

We added clarifications in the main text that better explain the advantage of the tool. The main contribution of this tool, specified in the Introduction, is that, unlike previous approaches for allosteric regulation, it combines all advantages of optogenetics (including regulation at a subcellular resolution) and enables broad applicability. We also further discussed the advantages of LightR as an allosteric activation tool in the Discussion section and dedicated a paragraph to comparing this method to other optogenetic tools enabling direct regulation of protein kinase activity.

6) Modulation of CRE by LightR is not mentioned in the Discussion. Moreover, there have been several reports where CRE activity (split strategy) has been utilized to control its activity (PMID: 32709899; PMID: 32358538; PMID: 27723747; PMID: 27065233, including work where CRE is regulated by VVD dimerization (e.g. Sheets et al., 2020 and Kennedy et al., 2010). Those examples should be referenced, and the advantage of having a light-activated CRE in only one construct, as opposed to two (split strategy) should be mentioned.

To address this comment, we dedicated a paragraph in the Discussion section of the revised manuscript to compare LightR-Cre with other split optogenetic Cre tools.

7) The work does not give a good sense of whether (or not) insertion of the tandem VVD domain perturbs kinase function. For example, the domain insertions in (Lee et al. (2008) Science 322:438) result in a dramatic decrease in the WT enzyme activity. The authors might address this by comparing their in vivo measurements to existing data for activated native Src or bRaf. Another method may be using in vitro kinase assays to directly measure activity in lit and dark states. If these experiments cannot be currently conducted, the authors should at least clearly discuss this point.

This is a great point. To address this, we added a panel in Figure 2A that shows results of an in vitro kinase assay for LightR-Src and constitutively active Src. These results demonstrate that the insertion of LightR domain in Src kinase completely abolishes its activity in the dark and restores it in the light to levels comparable to that of constitutively active Src.

8) "This indicates that the open conformation of LightR clamp in the dark can cause changes in the catalytic domain of Src that will be reversed upon illumination with blue light". The authors should also discuss if this approach could be implemented (by changing the type of linker, or where LightR is inserted) such that the "distorted" conformation can be reached in the light state, whereas the "WT/active" one could be achieved in the dark.

The description of the LightR should briefly mention the size of the utilized VVD (compared to what has been used in optogenetic systems), and the type of linker that was added in between both VVDs. This info is extractable from the figures, but it would help if it is explicitly mentioned: for example, how many different GS linker sizes were simulated in silico, and how many were experimentally tried before defining the definitive LightR construct.

To address this comment, we discussed a possibility to change the directionality of LightR regulation by changing the linkers or the insertion site of LightR (Discussion section).

We also indicated the size of LightR domain (335 amino acid total size) and specified the linker that was used between VVD domains. We simulated and tested only one linker. The linkers connecting LightR domain to the catalytic domain were selected based on previous studies (PMID: 20581846 and PMID: 24609359).

The GS-repeat linker between VVD domains was selected to provide sufficient length (approximately 25-30 Å) to allow dissociation and association of VVD monomers. This is now clarified in the revised version.

Author details

1. Mark Shaaya

   Department of Pharmacology and Regenerative Medicine, The University of Illinois at Chicago, College of Medicine, Chicago, United States

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4066-2693

2. Jordan Fauser

   Department of Pharmacology and Regenerative Medicine, The University of Illinois at Chicago, College of Medicine, Chicago, United States

   Contribution

   Formal analysis, Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0675-8977

3. Anastasia Zhurikhina

   Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, United States

   Contribution

   Software, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Jason E Conage-Pough

   1. The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States
   2. Center for Precision Cancer Medicine, Massachusetts Institute of Technology, Cambridge, United States
   3. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, United States

   Contribution

   Formal analysis, Investigation, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1614-9374

5. Vincent Huyot

   Department of Pharmacology and Regenerative Medicine, The University of Illinois at Chicago, College of Medicine, Chicago, United States

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

6. Martin Brennan

   Department of Pharmacology and Regenerative Medicine, The University of Illinois at Chicago, College of Medicine, Chicago, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

7. Cameron T Flower

   1. The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States
   2. Center for Precision Cancer Medicine, Massachusetts Institute of Technology, Cambridge, United States
   3. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, United States
   4. Program in Computational and Systems Biology, Massachusetts Institute of Technology, Cambridge, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9632-9913

8. Jacob Matsche

   Department of Pharmacology and Regenerative Medicine, The University of Illinois at Chicago, College of Medicine, Chicago, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

9. Shahzeb Khan

   Department of Pharmacology and Regenerative Medicine, The University of Illinois at Chicago, College of Medicine, Chicago, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

10. Viswanathan Natarajan

    Department of Pharmacology and Regenerative Medicine, The University of Illinois at Chicago, College of Medicine, Chicago, United States

    Contribution

    Supervision, Funding acquisition, Writing - review and editing

    Competing interests

    No competing interests declared

11. Jalees Rehman

    1. Department of Pharmacology and Regenerative Medicine, The University of Illinois at Chicago, College of Medicine, Chicago, United States
    2. University of Illinois Cancer Center, The University of Illinois at Chicago, Chicago, United States
    3. Division of Cardiology, Department of Medicine, The University of Illinois, College of Medicine, Chicago, United States

    Contribution

    Funding acquisition, Writing - review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-2787-9292

12. Pradeep Kota

    Marsico Lung Institute, Cystic Fibrosis Center and Department of Medicine, University of North Carolina, Chapel Hill, United States

    Contribution

    Formal analysis, Supervision, Funding acquisition, Investigation, Writing - review and editing

    Competing interests

    No competing interests declared

13. Forest M White

    1. The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, United States
    2. Center for Precision Cancer Medicine, Massachusetts Institute of Technology, Cambridge, United States
    3. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, United States
    4. Program in Computational and Systems Biology, Massachusetts Institute of Technology, Cambridge, United States

    Contribution

    Supervision, Funding acquisition, Writing - review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-1545-1651

14. Denis Tsygankov

    Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, United States

    Contribution

    Formal analysis, Supervision, Funding acquisition, Methodology

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-1180-3584

15. Andrei V Karginov

    1. Department of Pharmacology and Regenerative Medicine, The University of Illinois at Chicago, College of Medicine, Chicago, United States
    2. University of Illinois Cancer Center, The University of Illinois at Chicago, Chicago, United States

    Contribution

    Conceptualization, Supervision, Funding acquisition, Project administration, Writing - review and editing

    For correspondence

    karginov@uic.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-2370-6383

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