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    • The PDZ domains of DLG interact with tumor suppressor

    2023-02-10

    The PDZ domains of DLG interact with tumor suppressor proteins, APC and PTEN, as well as with several viral oncoproteins such as the E6 protein present in oncogenic human papillomavirus (reviewed in [177]). It has been reported that overexpression of DLG in fibroblasts impairs the events in the G0/G1 to S phase and inhibits cell proliferation [178]. DLG, like SCRIB, is therefore likely to be involved in the negative regulation of cell proliferation [177]. The interaction between APC and DLG regulated by PAR complex is involved in polarized cell migration [179]. Mammalian LGL proteins are linked to the exocytic machinery by direct interactions with SNARE proteins such as Syntaxin [125], [180], [181]. Zhang et al. [181] proposed that interactions between LGL proteins and the exocyst are important for the establishment and reinforcement of cell polarity. LGL1 is also associated with non-muscle heavy chain myosin II in a cytoskeletal network [165].
    Acknowledgements
    Introduction
    Genetically Encoded Reporters of Protein Kinases The discovery of the GFP and its engineering into a wide variety of AFPs prompted the development of GFP-based reporters and the design of genetically encoded biosensors, which were rapidly applied to the field of protein kinases.32, 33, 34, 35, 36, 39, 40, 41, 42, 43, 44, 45 Several groups of genetically encoded biosensors have been developed (for review, see Refs. 34,44): (i) single-chain biosensors, which bear a pair of FRET AFPs within the same molecule that are brought together and undergo energy transfer due to intramolecular conformational changes in response to the recognition event; (ii) two-chain biosensors, in which two AFPs lie on two different molecules capable of interacting and undergoing intermolecular FRET when the two chains are brought together for a protein/protein interaction; (iii) biosensors based on bimolecular fluorescence complementation (BiFC), in which two fragments of a split AFP are brought together as a consequence of the recognition event, thereby reconstituting the intact and fully fluorescent protein; (iv) single-chain biosensors which bear a single AFP whose spectral properties change in response to the recognition of a target by an encoded Vildagliptin other than the AFP; and (v) biosensors constituted by the AFP itself, whose spectral properties respond directly to the target or analyte, as pH-dependent or redox-dependent AFP biosensors (Fig. 6.2). The most widely developed genetically encoded biosensors developed to probe protein kinase activities are single-chain FRET biosensors that report on kinase activity through phosphorylation-induced changes in FRET between two AFPs due to an intramolecular conformational change. A wide variety of these genetically encoded biosensors have been developed (see Table 6.2 and Figure 6.3, Figure 6.4, Figure 6.5, Figure 6.6), but the basic structure of these kinase activity reporters (KARs) is essentially the same: a kinase-specific substrate sequence bearing a consensus phosphorylation site connected to a matching phosphoamino acid-binding domain (PAABD) by a flexible linker and flanked by a pair of AFPs that can transfer fluorescence resonance energy between each other. Following phosphorylation of the substrate sequence by the kinase, the PAABD binds the phosphorylated sequence, thereby promoting an intramolecular conformational change which alters the distance and orientation of the AFPs. Several factors have to be considered when aiming to design an optimally responsive and selective genetically encoded FRET biosensor. Moreover, the specificity and selectivity of a kinase/biosensor couple should always be characterized in vitro and in cellulo. First, needless to say, the size and sequence of the substrate have to be tailored to the most suitable sequence to gain the highest level of selectivity. The choice of the substrate sequence is essential for selectivity and may either be identified through an in silico approach through database mining from knowledge-based libraries, or simply through rational design of a sequence derived from a known substrate protein of a given kinase. The specificity of a kinase for a protein substrate is generally dependent on other regions of the protein that “dock” onto the kinase to offer higher affinity/recognition. Therefore, several designs include a docking domain distinct from the substrate sequence, so as to increase specificity for the target kinase while also increasing the efficiency of phosphorylation of the substrate by the kinase. This is well exemplified by MAPK protein kinases (ERK, p38, and JNK), which have very similar phosphorylation sites, yet distinct docking sites that can be made use of to improve substrate targeting and selectivity.81, 82