Characterization of the Intramolecular Interactions and Regulatory Mechanisms of the Scaffold Protein Tks4.

The scaffold protein Tks4 is a member of the p47phox-related organizer superfamily. It plays a key role in cell motility by being essential for the formation of podosomes and invadopodia. In addition, Tks4 is involved in the epidermal growth factor (EGF) signaling pathway, in which EGF induces the translocation of Tks4 from the cytoplasm to the plasma membrane. The evolutionarily-related protein p47phox and Tks4 share many similarities in their N-terminal region: a phosphoinositide-binding PX domain is followed by two SH3 domains (so called "tandem SH3") and a proline-rich region (PRR). In p47phox, the PRR is followed by a relatively short, disordered C-terminal tail region containing multiple phosphorylation sites. These play a key role in the regulation of the protein. In Tks4, the PRR is followed by a third and a fourth SH3 domain connected by a long (~420 residues) unstructured region. In p47phox, the tandem SH3 domain binds the PRR while the first SH3 domain interacts with the PX domain, thereby preventing its binding to the membrane. Based on the conserved structural features of p47phox and Tks4 and the fact that an intramolecular interaction between the third SH3 and the PX domains of Tks4 has already been reported, we hypothesized that Tks4 is similarly regulated by autoinhibition. In this study, we showed, via fluorescence-based titrations, MST, ITC, and SAXS measurements, that the tandem SH3 domain of Tks4 binds the PRR and that the PX domain interacts with the third SH3 domain. We also investigated a phosphomimicking Thr-to-Glu point mutation in the PRR as a possible regulator of intramolecular interactions. Phosphatidylinositol-3-phosphate (PtdIns(3)P) was identified as the main binding partner of the PX domain via lipid-binding assays. In truncated Tks4 fragments, the presence of the tandem SH3, together with the PRR, reduced PtdIns(3)P binding, while the presence of the third SH3 domain led to complete inhibition.

Structural Reconstruction of Protein-Protein Complexes Involved in Intracellular Signaling.

Signaling complexes within the cell convert extracellular cues into physiological outcomes. Their assembly involves signaling enzymes, allosteric regulators and scaffold proteins that often contain long stretches of disordered protein regions, display multi-domain architectures, and binding affinity between individual components is low. These features are indispensable for their central roles as dynamic information processing hubs, on the other hand they also make reconstruction of structurally homogeneous complex samples highly challenging. In this present chapter we discuss protein machinery which influences extracellular signal reception, intracellular pathway activity, and cytoskeletal or transcriptional activity.

Structural insights into the pSer/pThr dependent regulation of the SHP2 tyrosine phosphatase in insulin and CD28 signaling.

Serine/threonine phosphorylation of insulin receptor substrate (IRS) proteins is well known to modulate insulin signaling. However, the molecular details of this process have mostly been elusive. While exploring the role of phosphoserines, we have detected a direct link between Tyr-flanking Ser/Thr phosphorylation sites and regulation of specific phosphotyrosine phosphatases. Here we present a concise structural study on how the activity of SHP2 phosphatase is controlled by an asymmetric, dual phosphorylation of its substrates. The structure of SHP2 has been determined with three different substrate peptides, unveiling the versatile and highly dynamic nature of substrate recruitment. What is more, the relatively stable pre-catalytic state of SHP2 could potentially be useful for inhibitor design. Our findings not only show an unusual dependence of SHP2 catalytic activity on Ser/Thr phosphorylation sites in IRS1 and CD28, but also suggest a negative regulatory mechanism that may also apply to other tyrosine kinase pathways as well.

A non-catalytic herpesviral protein reconfigures ERK-RSK signaling by targeting kinase docking systems in the host.

The Kaposi's sarcoma associated herpesvirus protein ORF45 binds the extracellular signal-regulated kinase (ERK) and the p90 Ribosomal S6 kinase (RSK). ORF45 was shown to be a kinase activator in cells but a kinase inhibitor in vitro, and its effects on the ERK-RSK complex are unknown. Here, we demonstrate that ORF45 binds ERK and RSK using optimized linear binding motifs. The crystal structure of the ORF45-ERK2 complex shows how kinase docking motifs recognize the activated form of ERK. The crystal structure of the ORF45-RSK2 complex reveals an AGC kinase docking system, for which we provide evidence that it is functional in the host. We find that ORF45 manipulates ERK-RSK signaling by favoring the formation of a complex, in which activated kinases are better protected from phosphatases and docking motif-independent RSK substrate phosphorylation is selectively up-regulated. As such, our data suggest that ORF45 interferes with the natural design of kinase docking systems in the host.

MAP Kinase-Mediated Activation of RSK1 and MK2 Substrate Kinases.

Mitogen-activated protein kinases (MAPKs) control essential eukaryotic signaling pathways. While much has been learned about MAPK activation, much less is known about substrate recruitment and specificity. MAPK substrates may be other kinases that are crucial to promote a further diversification of the signaling outcomes. Here, we used a variety of molecular and cellular tools to investigate the recruitment of two substrate kinases, RSK1 and MK2, to three MAPKs (ERK2, p38α, and ERK5). Unexpectedly, we identified that kinase heterodimers form structurally and functionally distinct complexes depending on the activation state of the MAPK. These may be incompatible with downstream signaling, but naturally they may also form structures that are compatible with the phosphorylation of the downstream kinase at the activation loop, or alternatively at other allosteric sites. Furthermore, we show that small-molecule inhibitors may affect the quaternary arrangement of kinase heterodimers and thus influence downstream signaling in a specific manner.

Co-regulation of the transcription controlling ATF2 phosphoswitch by JNK and p38.

Transcription factor phosphorylation at specific sites often activates gene expression, but how environmental cues quantitatively control transcription is not well-understood. Activating protein 1 transcription factors are phosphorylated by mitogen-activated protein kinases (MAPK) in their transactivation domains (TAD) at so-called phosphoswitches, which are a hallmark in response to growth factors, cytokines or stress. We show that the ATF2 TAD is controlled by functionally distinct signaling pathways (JNK and p38) through structurally different MAPK binding sites. Moreover, JNK mediated phosphorylation at an evolutionarily more recent site diminishes p38 binding and made the phosphoswitch differently sensitive to JNK and p38 in vertebrates. Structures of MAPK-TAD complexes and mechanistic modeling of ATF2 TAD phosphorylation in cells suggest that kinase binding motifs and phosphorylation sites line up to maximize MAPK based co-regulation. This study shows how the activity of an ancient transcription controlling phosphoswitch became dependent on the relative flux of upstream signals.