Kinase activity-independent regulation of cyclin pathway by GRK2 is essential for zebrafish early development.
G protein-coupled receptor (GPCR) kinases (GRKs) are known as a family of serine/threonine kinases that function as key regulators of GPCRs, as well as other types of receptors. Extensive studies of GRKs at the cellular and organismal levels have led to a consensus that GRK-catalyzed phosphorylation of receptors is the primary mechanism underlying their physiological functions. Here, we report that down-regulation of GRK2 in zebrafish embryos with GRK2 morpholino results in developmental early arrest and, interestingly, that this arrest can be rescued by exogenous expression of a GRK2 kinase-dead mutant, K220R. A physical interaction between GRK2 and cyclin B1 regulator patched homolog 1 (PTCH1), stimulated by Hedgehog (Hh), rather than GRK2-mediated phosphorylation of downstream targets, appears as the underlying mechanism. We identify residues 262-379 as the PTCH1-binding region (BP). Interaction of GRK2, K220R, and BP with PTCH1 reduces the association of PTCH1 with cyclin B1 and disrupts PTCH1-mediated inhibition of cyclin B1 nuclear translocation, whereas the PTCH1-binding deficient GRK2 mutant (Delta312-379) does not. Cell cycle and cell proliferation assays show that overexpressing PTCH1 remarkably inhibited cell growth and this effect could be attenuated by GRK2, K220R, or BP, but not Delta312-379. In vivo studies show that BP, as well as the nuclear-localizing cyclin B1 mutant, is effective in rescuing the early arrest phenotype in GRK2 knockdown embryos, but Delta312-379 is not. Our data thus reveal a novel kinase activity-independent function for GRK and establish a role for GRK2 as a cell-cycle regulator during early embryonic development.
Jiang
Xi
X
State Key Laboratory of Medical Neurobiology and Pharmacology Research Center, Shanghai Medical College and Institutes of Brain Science, Fudan University, Shanghai, China.
Yang
Peng
P
Ma
Lan
L
D000818:Animals; D002417:Cattle; D049109:Cell Proliferation; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D016213:Cyclins / Q000378:metabolism*; D015536:Down-Regulation; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins; D009154:Mutation; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D011956:Receptors, Cell Surface / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D015027:Zebrafish / Q000196:embryology* / Q000378:metabolism; D029961:Zebrafish Proteins
C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D016213:Cyclins; D008565:Membrane Proteins; C000606236:PTCH1 protein, human; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D011956:Receptors, Cell Surface; D029961:Zebrafish Proteins; C491715:ptch1 protein, zebrafish; D054769:G-Protein-Coupled Receptor Kinase 2