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.

PKA is a holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. There are two major families of C-subunits, C and C, and four functionally nonredundant R-subunits (RI, RI, RII, RII). In addition to binding to and being regulated by the R-subunits, the C-subunits are regulated by two tails-regions that each wrap around the N-and C-lobes of the kinase core. While the Ct-Tail is classified as an intrinsically disordered region (IDR), the Nt-Tail is dominated by a strong helix that is flanked by short IDRs. In contrast to the Ct-Tail, which is a conserved and highly regulated feature of all AGC kinases, the Nt-Tail has evolved more recently and is highly variable in vertebrates. Surprisingly and in contrast to the kinase core and the Ct-Tail, the entire Nt-tail is not conserved in non-mammalian PKAs. In particular, in humans C actually represents a large family of C-subunits that are highly variable in their Nt-Tail and also expressed in a highly tissue-specific manner. While we know so much about the C1 subunit, we know almost nothing about these C isoforms where C2 is highly expressed in lymphocytes and C3 and C4 isoforms account for ~50% of PKA signaling in brain. Based on recent disease mutations, the C proteins appear to be functionally important and non-redundant with the C isoforms. Imaging in retina also supports non-redundant roles for C as well as isoform-specific localization to mitochondria. This represents a new frontier in PKA signaling.

Protein kinase have evolved to be dynamic macromolecular switches that alternate between inactive and active conformations. Although most kinase are typically phosphorylated and activated by other kinases, relatively little is known about the detailed mechanistic steps required for one kinase to activate another. The p90 ribosomal S6 kinases (RSKs) lie downstream of the Ras‐MAPK pathway and regulates cell proliferation, cell survival, cell growth, and cell motility. RSK is an interesting model system to study kinase activation because it contains two distinct kinase domains, an N‐terminal kinase (NTK), and a C‐terminal kinase (CTK) in the same polypeptide chain. RSK has a complex activation mechanism that includes sequential phosphorylation events and requires two additional kinases: extracellular signal‐regulated kinase (ERK) and 3‐phosphoinositide‐dependent protein kinase 1 (PDK1). The precise structural details of RSK kinase activation are not fully understood and a more rigorous structural characterization could provide valuable insight into how kinase complexes assemble and how kinases activate other kinases. RSK activation begins with ERK binding to and activating the CTK. A major goal of this project is to determine the structure of the full length RSK‐ERK complex primarily utilizing cryoEM to gain insight into the orientations of three kinase domains relative to one another and to identify specific interaction networks that exist within the complex. We have determined a low resolution density map of the RSK‐ERK complex using negative stain EM and can identify each kinase domain.

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Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline mutations in the diacylglycerol (DG)/Ca2+‐regulated protein kinase C gamma (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. Curiously, the majority of the approximately 50 missense mutations identified in PKCγ cluster to the DG‐sensing C1A and C1B domains. Here, we use a genetically‐encoded FRET‐based C Kinase Activity Reporter (CKAR) to show that ataxia‐associated PKCγ mutants have higher basal activity in cells, and thus are less autoinhibited, than wild‐type enzyme. However, whereas reduced autoinhibition generally renders PKC sensitive to degradation, we show that mutations in the C1B domain allow translocation to membranes but protect PKCγ from phorbol ester‐induced down‐regulation. Indeed, deletion of the C1B domain prevents PKCγ down‐regulation with phorbol esters, potent ligands for the C1 domains. Strikingly, the degree of impaired autoinhibition correlates inversely with age of disease onset. Patients with the most severe mutation we examined (V138E) present with symptoms as young children, whereas symptoms in patients with the least severe mutation examined (D115Y) manifested in their 40s. To understand the structural basis of mutations outside the C1 domains, we generated a model of PKCγ using homology modeling and molecular docking. Mutations outside the C1 domains occur in regions also predicted to disrupt autoinhibition, including the pseudosubstrate, a predicted interface between the kinase and C1B domains, and the C‐terminal tail of PKCγ. Taken together, our data support a model in which SCA14 mutations enhance PKCγ activity without compromising stability. Furthermore, because many of the genetic causes of the 40+ types of SCA alter Ca2+ homeostasis, deregulated PKCγ activity may be a common cause for the disease. This raises the possibility that inhibition of PKCγ will be a potentially viable therapeutic target for SCA. Support or Funding Information NIH T32 GM007752