PodcastPhosphoproteomics

Science Signaling Podcast: 18 August 2009

Sci. Signal.  18 Aug 2009:
Vol. 2, Issue 84, pp. pc15
DOI: 10.1126/scisignal.284pc15

Abstract

This is a conversation with David Han about a Research Article published in the 18 August 2009 issue of Science Signaling.

(Length: 19 min; file size: 8.96 MB; file format: mp3; location: http://podcasts.aaas.org/science_signaling/ScienceSignaling_090818.mp3)

Technical Details

Length: 19 min

File size: 8.96 MB

File Format: mp3

RSS Feed: http://stke.sciencemag.org/rss/podcast.xml

Download Podcast: http://podcasts.aaas.org/science_signaling/ScienceSignaling_090818.mp3

Educational Details

Learning Resource Type: Audio

Context: High school upper division 11-12, undergraduate lower division 13-14, undergraduate upper division 15-16, graduate, professional, general public and informal education

Intended Users: Teacher, learner

Intended Educational Use: Learn, teach

Discipline: Biochemistry, Cell Biology, Immunology, Proteomics

Keywords: Science Signaling, phosphoproteomics, phosphorylation, protein-protein interaction, proteomics, systems biology, T cell activation, T cell receptor, TCR

Transcript

Host – Annalisa VanHookWelcome to the Science Signaling Podcast for August 18th, 2009. I’m Annalisa VanHook. In this episode, I'm speaking with David Han from the University of Connecticut about a Research Article from his group published in the current issue of Science Signaling (1). The paper describes their research on phosphorylation events downstream of T cell receptor activation. T cell receptor signaling is crucial for the activation of T cells, an important event in mounting an immune response to combat pathogens. Dr. Han spoke with me on the phone from his lab in Farmington, Connecticut.

Interviewer – Annalisa VanHookHello, Dr. Han.

Interviewee – David HanHello.

Interviewer – Annalisa VanHookBefore we talk about the specific findings of your current paper, can you give us a little background on T cell signaling – what the T cell receptor is and what it does?

Interviewee – David HanSure. T cell receptor is the main receptor that sense antigens, so a T cell receptor typically is the αβ receptor, which is a cell surface dimeric protein. It doesn’t have any inherent cytosolic kinase domain, but it associate[s] with a number of other proteins, such as CD3 protein complexes, which are α, γ, δ, ε, and ζ proteins. And essentially what T cell receptor does is when it encounters foreign antigen [is] presented by antigen-presenting cell, if it is [a] specific antigen that it recognizes, T cell will go through series of steps to activate itself, and this process is integral to immunity, in defense against viruses, bacteria—it’s mediated by this receptor, T cell receptor. So we study this process—this T cell receptor signaling—how T cell receptor sense and communicate to the T cell to go undergo a series of activation steps.

Interviewer – Annalisa VanHookSo, despite the fact that the T cell receptor itself does not have kinase activity, there are a lot of protein phosphorylation events that happen downstream of T cell receptor activation. As I understand, these are the proteins that your current paper focused on?

Interviewee – David HanYes. We are interested in how phosphorylation, which is one of the many ways that the cell utilize to transmit signaling, orchestrate many diverse cellular processes. And we use this T cell receptor model system because this is a system that’s been studied for the last 20 years or so. And, as you were saying, although T cell receptor does not have [an] intracellular kinase domain, it activates intracellular kinases such as Lck or ZAP70 family of tyrosine kinases, and it transmit phosphorylation cascade, and it’s known that phosphorylation cascade is essential and required for T cell activation. So, if you were to mutate Lck, or ZAP70, you don’t get full activation of T cell. So, phosphorylation cascade is one of the key steps required for full activation of T cell. So, what we did was, we studied phosphorylation event[s], not just tyrosine but global phosphorylation event[s], using proteomics in this in this paper.

Interviewer – Annalisa VanHookCould you briefly explain the, the approach you took to studying these phosphorylation events?

Interviewee – David HanWe took the system-level approach. In the system-level approach, the assumption is not just looking at a few signaling events, but looking at hundreds or thousands of signaling events experimentally at the same time, followed by using these experimental measurements to distill it down to a coherent picture, will likely give us new signal transduction insights. So, we utilized this phosphoproteomic approach, which allow us to capture thousands and thousands of phosphorylation sites at the same time. So, what the experiment we did was, we took either unstimulated or stimulated T cells and captured phosphopeptides using proteomic approaches, and we quantify how many of those are changing during T cell activation, as well. So, putting this wide net allow us to capture over 10,000 phosphorylation sites, among which about 690 or so are inducibly changing during T cell activation.

Interviewer – Annalisa VanHookWere you looking at all the phosphorylation sites in a cell, or were you looking only at tyrosine phosphorylation sites or only at serine phosphorylation sites?

Interviewee – David HanThat’s a good question. We were looking at both. And, you’re right that, you know, early studies in T cells have been focusing mainly on tyrosine phosphorylation—this is in part due to the availability of antiphosphotyrosine antibodies and other affinity reagents that allow people to capture tyrosine phosphorylated proteins and substrates. But in terms of T cell receptor signaling, very few serine-threonine phosphorylation events have been characterized—there are some but not a lot. But, in contrast, if you look in the cell and ask the question, “What percent of phosphoproteins are phosphorylated on tyrosine versus serine-threonine?” Less than 1% of phosphorylation events are on tyrosine, and a large majority of the phosphorylation events are on serine and threonine, although very little has been characterized in terms of their exact function in T cell activation. So, it turned out that our paper focuses more on serine-threonine phosphorylation because we capture, you know, out of 10,000 or so, over 90% of those turn out to be on serine and threonine residues.

Interviewer – Annalisa VanHookYou weren’t looking at individual proteins—you were looking at phosphorylation sites—so, one protein might have four phosphorylation sites, for example.

Interviewee – David HanCorrect. Although we identified over 10,000 unique sites, those represent only about 3,000 proteins. Like you said, there are proteins with multiple phosphorylation sites.

Interviewer – Annalisa VanHookSo, when you started looking at all of the phosphorylation sites and all the proteins that you identified from the screen, what sort of themes emerged? Did any groups of functionally similar proteins become apparent, or could these proteins be grouped by biochemical function or by where they are in a cell?

Interviewee – David HanYes. That’s a very, very good question. First of all, I must say that we there is a general theme that emerge from this screen, and we would not be able to make that conclusion without many contributions from seminal work from many investigator[s] over the last 20 years in T cell field. This is a field where people have characterized—for example, when T cells get activated they form a so-called immunological synapse. That is, [a] T cell and [an] antigen-presenting cell form [an] adhesive interface where receptors are organized into discrete layers. T cell receptors organize in the center area, and there are other receptors that are surrounding as rings of receptors, and these are to insure that T cell fully interact and engage and interpret antigen for a prolonged period of time so that T cell can undergo fully [the] activation process: T cell can undergo a so-called clonal expansion where it start to proliferate; it can secrete cytokines to the neighboring cells as well as to the antigen-presenting cells and B cells, for example, to start making antibodies.

So, this process has been very well characterized, and there are several major phenomena that’s been described during T cell activation. One of it is so-called cell surface protein patterning—organization of receptors into discrete layers. This again is actin cup formation—there is actin polymerization right at the junction, immunological synapse area. And then there are, too, microtubule polarization where microtubule is utilized to recruit cytokines or endocytosis process, so microtubule organization is dramatically changed. And this process occur[s] within a few minutes and continue on for a prolonged period of time after T cell activation. So, looking at well characterized biological processes and then looking at experimental data of the proteins belonging to these functional modules, such as receptor patterning, actin, tubulin, transcription modules, we were able to deduce a coherent theme—that is, how phosphorylation could potentially mediate these processes. So, the emergent theme is that phosphorylation targets all these functional modules, and these—each module may have 20, 30 proteins—and those modules are extensively and inducibly phosphorylated.

And it turn out that when you look at the actual sites of phosphorylation, those sites turn out to be on protein-protein interaction domains. And when we looked at all these modules, as well as literature, there’s already 34 characterized sites that have people have shown that these phosphorylation sites on serine and threonine residues, either bring together two proteins or disrupt protein-protein interaction. So, that’s the emergent theme that come out of this study is that phosphorylation on serine-threonine targets protein modules on the protein-protein interaction sites, and, as a result of phosphorylation, proteins can either come together or disrupt association.

Interviewer – Annalisa VanHookMost of, or a good chunk of, the phosphorylation sites that you identified as being modified in response to activation of the T cell, regulated protein-protein interactions rather than regulating the enzymatic activity of a protein.

Interviewee – David HanThat’s correct. We were able to assign about 96 phosphorylation events that, for example, in—you know, we didn’t measure any activity per se, enzymatic activity, but we looked at protein-protein interactions and phosphorylation. It’s known that phosphorylation also modulate enzymatic activities. In [the] case of kinases, for example, it’s very well characterized. But, our focus in this study is mainly on protein-protein interaction.

Interviewer – Annalisa VanHookSo, it isn’t necessarily that phosphorylation events downstream of T cell receptor activation don’t regulate the catalytic activity of proteins, it’s just that in this study you focused on the protein-protein interactions, rather than on the catalytic activities.

Interviewee – David HanCorrect. But, it’s not necessarily mutually exclusive either, where, you know, protein-protein interaction can either promote or reduce catalytic activity. Another emerging theme is that phosphorylation cause intramolecular or intermolecular protein-protein interaction changes. A lot of time a protein, when it’s phosphorylated it may actually bind to itself, which may expose the catalytic domain, for example.

You know, the reason we are so excited about this study is that people know that, for example, tyrosine phosphorylation recruit[s] proteins through SH2 domain[s], and it’s not new—it’s been very well characterized. Even for serine-threonine phosphorylation, there are 14-3-3 kind of domains—proteins that interact with phosphoserine or phosphothreonine is well known. One point that we’re very excited about is the extent and the scope of phosphorylation where the whole protein module, such as actin remodeling module where F-actin bundling or branching or polymerization—all these proteins are targeted. And when you look at these proteins, the exact site of phosphorylation sites are on protein-protein interaction sites. And we know the F-actin cup is rapidly formed, so that allow us to deduce that phosphorylation is targeting specifically F-actin in all these proteins and bringing together or disrupting protein-protein interactions.

Second point that we’re very excited about is that most studies know that initial receptor activation results in some kind of kinase activation, which somehow transmit all throughout the cytoplasm into the nucleus. But, if you look at protein concentration inside the cell, it’s very, very high to the level of crystallographic protein concentration, with many, many proteins with different abundances all mixed together. So, the question is, “How can you achieve specific high-affinity protein interaction?” And it cannot be static—you know, proteins combine to another protein and that—if it is static—that’s not signal transduction. There has to be, those complexes has to be regulated precisely where it comes and bind and complex also have to break apart so that signals can flow from one protein to the next to the next to the next.

So, what we’re excited about is that for the first time we can begin to see—this is, again, it's a very first step—where we can see that a number of phosphorylation events are targeting protein-protein interaction domains. And secondly, it does not only bring together proteins, it can also disrupt complexes, so serine-threonine phosphorylation being the predominant force in the cell, so as you can see serine wave or serine-threonine phosphorylation occurs throughout the cell, you start to imagine complexes forming and complexes disruption, which cause, like you said, activity changes, localization changes, and that’s the basis—one basis—of signal transduction. That’s why we’re very excited about it. And there's also the third reason why we excited about is—you know, if you look at a number of protein complexes, such as proteins that cause lymphoma, leukemia, those kind of complexes, those proteins are heavily phosphorylated. We found about 100 or 170 proteins, and there are close to 900 phosphorylation sites. The possibility that all of these phosphorylation sites may be interaction domains, and depending on phosphorylated version or not phosphorylated version, one can potentially have different associations, different activities—so that becomes a very exciting point for us that we’re beginning to see how complex the signal transduction is.

Interviewer – Annalisa VanHookNow that you’ve got this large list of protein phosphorylation sites that are affected downstream of T cell activation, what next? What do you do with this big list of proteins?

Interviewee – David HanI think it is it would be very useful for many people studying in T cell biology field—so, there are people dedicating their life to study a few proteins, you know, really understand the intricacies of how this protein is involved in T cell activation. And we now have specific sites that—for example, one of the proteins called BCL11B, the phosphorylation has not been characterized for this protein, and now we show multiple phosphorylation sites on this protein. It’s a very important protein, [a] transcription factor that interact with transcriptional repressor, binds to DNA to activate il-2 gene. Now, we came up with a hypothesis of how BCL11B phosphorylation relieved this protein from a repressive state and allows it to bind and activate il-2 promoter and make mRNA transcript. Now, of course this is still a hypothesis, but a couple of groups studying BCL11B, and I’m sure they will be very excited to look at that and further validate whether our hypothesis is correct or not.

Similarly, there are many investigators interested in T cell or just in general field of signal transduction, people are interested in actin polymerization, people are interested in microtubule[s] and how microtubules can be modulated so that you get selective microtubule polymerization at selected sites of the cell. Those investigators will be very interested in the data that we have. And, you know, in terms of disease standpoint, people who are interested in targeting specific kinases, such as BCR-ABL, using specific kinase inhibitor, they can look at the network that we’ve come up with, where we integrated protein-protein interaction with phosphorylation. And you begin to see there are multiple proteins in this network potentially modulated by phosphorylation so that if pharmaceutical companies are thinking of designing drugs to inhibit one kinase, I would say, instead of just targeting one kinase in a pathway, now you can begin to look at targeting multiple kinases or multiple proteins in this network. So, those kind of things that one begin to think about. But, of course a lot of work needs to be done.

Interviewer – Annalisa VanHookThank you, Dr. Han, for speaking with me.

Interviewee – David HanThank you very much—really appreciate it.

Host – Annalisa VanHookThat was David Han discussing a Research Article published in the August 18th issue of Science Signaling. The article is by Mayya and colleagues and is titled, “Quantitative Phosphoproteomic Analysis of T Cell Receptor Signaling Reveals System-Wide Modulation of Protein-Protein Interactions” (1).

music

And that wraps up this Science Signaling Podcast. If you have any questions or suggestions, please write to us at sciencesignalingeditors{at}aaas.org. This show is a production of Science Signaling and of AAAS—Advancing Science, Serving Society. I'm Annalisa VanHook, and on behalf of Science Signaling and its publisher, the American Association for the Advancement of Science, thanks for listening.

Reference

View Abstract

Navigate This Article