Carol MacKintosh

The protein kinases PAK4, PAK5 and PAK6 comprise a family of ohnologues. In multiple cancers including melanomas PAK5 most frequently carries non-synonymous mutations; PAK6 and PAK4 have fewer; and PAK4 is often amplified. To help interpret these genomic data, initially we compared the cellular regulation of the sister kinases and their roles in melanoma cells. In common with many ohnologue protein kinases, PAK4, PAK5 and PAK6 each have two 14-3-3-binding phosphosites of which phosphoSer99 is conserved. PAK4 localises to the leading edge of cells in response to phorbol ester-stimulated binding of 14-3-3 to phosphoSer99 and phosphoSer181, which are phosphorylated by two different PKCs or PKDs. These phosphorylations of PAK4 are essential for its phorbol ester-stimulated phosphorylation of downstream substrates. In contrast, 14-3-3 interacts with PAK5 in response to phorbol ester-stimulated phosphorylation of Ser99 and epidermal growth factor-stimulated phosphorylation of Ser288; whereas PAK6 docks onto 14-3-3 and is prevented from localising to cell-cell junctions when Ser133 is phosphorylated in response to cAMP-elevating agents via PKA and insulin-like growth factor 1 via PKB/Akt. Silencing of PAK4 impairs viability, migration and invasive behaviour of melanoma cells carrying BRAF^V600E or NRAS^Q61Kmutations. These defects are rescued by ectopic expression of PAK4, more so by a 14-3-3-binding deficient PAK4, and barely by PAK5 or PAK6. Together these genomic, biochemical and cellular data suggest that the oncogenic properties of PAK4 are regulated by PKC–PKD signalling in melanoma, while PAK5 and PAK6 are dispensable in this cancer.

Dibble et al. (2022) find that insulin via PI 3-kinase–Akt signalling stimulates cellular synthesis of Coenzyme A (CoA). CoA is synthesised from pantothenate (vitamin B5), ATP and cysteine via a 5-step pathway starting with pantothenate kinase, which is catalysed by PANK1, 2 and 3. Moreover, PANK2 and the lesser-studied PANK4 undergo PI 3-kinase-dependent phosphorylations on Akt-consensus sites in cell cultures and in vivo in mice. Cellular expression of PANK4 suppresses CoA synthesis, whereas phosphorylation of Thr406 on PANK4 decreases this suppression. The mechanism involves a DUF89 phosphatase domain on PANK4 that dephosphorylates 4′-phosphopantetheine, and this phosphatase is inhibited by Thr406 phosphorylation of PANK4. Overall, in response to growth factors or oncogenes, Akt phosphorylates and inhibits PANK4, thereby promoting synthesis of CoA, which is needed for nutrient catabolism, lipid synthesis and proliferation.

Having discovered that ubiquitin-specific peptidase 18 (USP18) expression is increased in lung and other cancers, here the authors found that engineered reduction of USP18 expression in lung cancer cells decreased cell growth, wound-healing, migration, and invasion, and decreased lung cancer metastases. Moreover, USP18 knockdown caused a decrease in expression of 14-3-3ζ. 14-3-3ζ is one of the family of proteins that dock onto pairs of phosphosites on target proteins, including sites that are phosphorylated in response to oncogenic PI 3-kinase/PKB and Erk/p90Rsk signalling {1}. Experiments to knockdown and rescue 14-3-3ζ suggested that the actions of this protein contribute to lung cancer cell metastases. These findings provide a strong rationale for developing an inhibitor of the deubiquitinase USP18 to combat metastases.

There was an error in J. Cell Sci. (2012) 125, 4662-4675 (doi:10.1242/jcs.110296).A duplication of the bottom two GST blots in Fig. 2B occurred

Vertebrates emerged from their invertebrate ancestor via two rounds of whole-genome duplication (2R-WGD) over 500 mya. The extra sets of duplicate genes that remain have diversified to encode multistranded metabolic and regulatory networks that underpin vertebrate complexity and variety. The mechanisms of these ancient genome duplications had not been understood, and particularly whether they happened due to autopolyploidy or allopolyploidy. Autopolyploidy occurs due to duplication of the genome within a species whereas allopolyploidy results from hybridisation between two distinct but related species. This study by Simakov et al. compared chromosomes of a small marine invertebrate amphioxus to those of other invertebrates, the jawless lampreys, and a variety of jawed vertebrates. These high-resolution, data-enabled reconstructions of the 17 pairs of chromosomes in the pre-2R-WGD ancestor from which amphioxus gradually evolved. The authors were also able to deduce that one offspring of the ancestor experienced the first genome duplication, an autotetraploidy event. The resulting animal evolved into the lampreys, as well as another lineage that underwent a speciation event, generating distinct α and β animals. Down the line, the α and β animals came back together and hybridised in an allotetraploidy event, marking the second round of WGD. The resulting α/β hybrid was octoploid, except for chromosomal regions that had been differentially lost in α and β. This octoploid animal subsequently underwent further chromosomal losses, rearrangements culminating in rediploidisation and small-scale mutations to evolve into all the different lineages of jawed vertebrates. It's amazing to use data from modern-day animals to reconstruct events from half a billion years ago!

Diabetic cardiomyopathy is a progressive disease in diabetic patients, and myocardial insulin resistance contributes to its pathogenesis through incompletely-defined mechanisms. Striated muscle preferentially expressed protein kinase (SPEG) has two kinase-domains and is a critical cardiac regulator. Here we show that SPEG is phosphorylated on Ser²⁴⁶¹/Ser²⁴⁶²/Thr²⁴⁶³ by protein kinase B (PKB) in response to insulin. PKB-mediated phosphorylation of SPEG activates its second kinase-domain, which in turn phosphorylates sarcoplasmic/endoplasmic reticulum calcium-ATPase 2a (SERCA2a) and accelerates calcium re-uptake into the SR. Cardiac-specific deletion of PKBα/β or a high fat diet inhibits insulin-induced phosphorylation of SPEG and SERCA2a, prolongs SR re-uptake of calcium, and impairs cardiac function. Mice bearing a Speg^3A mutation to prevent its phosphorylation by PKB display cardiac dysfunction. Importantly, the Speg^3A mutation impairs SERCA2a phosphorylation and calcium re-uptake into the SR. Collectively, these data demonstrate that insulin resistance impairs this PKB-SPEG-SERCA2a signal axis, which contributes to the development of diabetic cardiomyopathy.

Ohnologs are genes in families generated by whole-genome duplications. They are named after Susumu Ohno who published his classic hypothesis of Evolution by Gene Duplication in 1970 {1}. Evolution of the vertebrates has been marked by two sequential whole-genome duplications (2R-WGD) at the invertebrate-to-vertebrate transition around 500 million years ago (Mya), followed by a teleost fish-specific 3R-WGD approximately 300 Mya, and a salmonid-specific WGD (4R) approximately 95 Mya. Interestingly, those ohnologs from the 2R, 3R and 4R that still survive in the genomes of modern-day vertebrates are highly enriched in components of signal transduction systems {2}. OHNOLOGS v2 is a database of ohnologs that stem from the 2R-WGD, which was compiled by comparative analysis of the genomes of 27 vertebrate species. Four of the species selected for analysis are fish, which also enabled tracking of ohnologs generated by the teleost fish-specific 3R-WGD. Thanks are due to the authors Param Priya Singh and Hervé Isambert for producing such a useful resource. OHNOLOGS v2 is easy to search, browse and download. No doubt it will be an invaluable tool for researching how vertebrate complexity and variety evolved across geologic time, and how cellular signal transduction networks operate in health and disease.

An evolutionary genetic analysis of the four vertebrate Robo receptor genes identifies neo-functionalisation of Robo4/Magic Roundabout, consistent with its coevolution within a wider signalling network that underpinned evolution of the vertebrate endothelium. The data indicate how a change in transcriptional regulation of Robo 4 in early vertebrate evolution may have set the stage for subsequent changes in the Robo4 protein towards its modern-day functions in modulating endothelial barrier function.