Application of Active and Kinase-Deficient Kinome Collection for Identification of Kinases Regulating Hedgehog Signaling

Abstract

To allow genome-scale identification of genes that regulate cellular signaling, we cloned >90% of all human full-length protein kinase cDNAs and constructed the corresponding kinase activity-deficient mutants. To establish the utility of this resource, we tested the effect of expression of the kinases on three different cellular signaling models. In all screens, many kinases had a modest but significant effect, apparently due to crosstalk between signaling pathways. However, the strongest effects were found with known regulators and novel components, such as MAP3K10 and DYRK2, which we identified in a mammalian Hedgehog (Hh) signaling screen. DYRK2 directly phosphorylated and induced the proteasome-dependent degradation of the key Hh pathway-regulated transcription factor, GLI2. MAP3K10, in turn, affected GLI2 indirectly by modulating the activity of DYRK2 and the known Hh pathway component, GSK3beta. Our results establish kinome expression screening as a highly effective way to identify physiological signaling pathway components and genes involved in pathological signaling crosstalk.

DYRK2 priming phosphorylation of c-Jun and c-Myc modulates cell cycle progression in human cancer cells

“…A recent report indicated that DYRK2 functions as a scaffold for an E3 ligase complex and controls mitotic transition (18). Another study demonstrated that DYRK2 contributes to proteasomal degradation of the transcription factor GLI2 (19). These findings shed light on a role for DYRK2 in protein proteolysis.…”

Activation of the hedgehog-signaling pathway in human cancer and the clinical implications

“…Gli molecules are now processed to active forms (GliA), which will activate the Hh target genes. (Evangelista et al, 2008;Varjosalo et al, 2008), but their exact functions in Hh signaling remain to be established.…”

Mechanism and evolution of cytosolic Hedgehog signal transduction

“…Whether loss of Fu activity is compensated for in vertebrate Hh signaling is unclear. It is also not known if other kinases such as Cdc2l1 (Cdk11b -Mouse Genome Informatics) or Ulk3 have replaced Fu in the mammalian Hh pathway, although an investigation of whether these kinases bind and phosphorylate Kif7 is warranted given their positive effects on Gli activity in vitro (Evangelista et al, 2008;Maloverjan et al, 2010;Varjosalo et al, 2008). In zebrafish, fu morphant phenotypes have revealed an Hh-independent role for fu in the biogenesis of motile cilia (Wilson et al, 2009a).…”