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  • G protein coupled receptor kinases GRKs are another group

    2021-11-26

    G protein-coupled receptor kinases (GRKs) are another group of kinases whose limited substrate repertoire is associated with an extensive binding interface. GRKs phosphorylate activated G protein-coupled receptors (GPCRs) at multiple sites, promoting binding of arrestin proteins to mediate receptor desensitization and G protein-independent signaling [32]. Both localization of GRKs to the plasma membrane, as well as direct physical interactions with the GPCR, contribute to specific targeting. In part because they form transient, state-dependent, low affinity complexes, structural insight into the nature of GRK–GPCR interactions has been limited. Recent studies using chemical crosslinking and hydrogen–deuterium exchange mass spectrometry have mapped points of interaction between GRK5 and its substrate β2-adrenergic receptor (B2AR) [33]. These studies suggest a structural model in which the GRK catalytic domain and its associated regulator of G-protein signaling homology (RH) domain directly contact multiple intracellular loops of the GPCR in addition to the C-terminal tail that harbors sites of phosphorylation. In addition to promoting recruitment of the kinase to its substrate, interaction with a GPCR also increases GRK catalytic activity. Prior crystallographic analyses of GRKs have suggested that the RH domain holds the catalytic domain in an open, inactive conformation [34]. Interaction with the B2AR appears to promote an elongated conformation in GRK5 in which specific autoinhibitory interactions between the RH and catalytic domains are broken. Activation of the kinase only upon direct binding to its substrate provides a mechanism for enforcing tight control of substrate specificity.
    Multisite Phosphorylation: Signal Integration, Amplification and Attenuation A large majority of phosphoproteins are phosphorylated at multiple sites, often by distinct protein kinases. While multiple kinases can phosphorylate a substrate independently of each other, multisite Birinapant can occur in a hierarchical manner, in which prior phosphorylation generates a recognition motif for a kinase to phosphorylate other sites [35]. This phenomenon of substrate ‘priming’ is a common mechanism for signal integration and amplification in eukaryotic signaling pathways [36]. Recent studies have expanded the scope of priming-dependent phosphorylation and provided insight into its structural basis. Classically, substrate priming occurs close to the site of subsequent phosphorylation. For some kinases, such as casein kinase 2, phosphorylated residues can substitute for negatively charged Asp or Glu residues found at multiple positions near its phosphorylation sites 18, 37 (Table 1). By contrast, glycogen synthase kinase 3β (GSK3β) is an obligate priming-dependent kinase with strict positional selectivity, requiring a phosphoserine (pSer) residue at the +4 position in its substrates (Table 1). Prior biochemical studies had suggested that the priming phosphate group binds GSK3β at a site analogous to that occupied by a phosphorylated residue within the activation loop of other kinases, leading to allosteric activation of the kinase [38]. This model has been confirmed through a series of X-ray cocrystal structures of GSK3 bound to peptides harboring pSer, which offered additional details of how priming phosphorylation promotes substrate binding [39]. While generally associated with STKs, in some cases TyrKs can also recognize primed substrates through catalytic site interactions 40, 41. Recent studies have investigated hierarchical phosphorylation by the TyrKs BMX [42] and epidermal growth factor receptor (EGFR) [43]. EGFR has a preference for a pTyr residue at the +1 position, mediating signal integration with the nonreceptor TyrK SRC through dual phosphorylation of the SHC adaptor protein. Cocrystal structures with EGFR revealed that primed substrates have a binding mode distinct from unprimed substrates: the +1 pTyr residue interacts with a Lys residue in the catalytic cleft that interacts with residues upstream of the phosphorylation site in nonprimed substrates. These observations suggest the overall EGFR recognition motif may differ between primed and unprimed substrates, or that substrate priming may override the necessity for a canonical consensus sequence. As the analogous catalytic cleft residue is either Lys or Arg in all human receptor TyrKs, priming-dependent phosphorylation may prove to be a general phenomenon among kinases in this group.