G protein-coupled receptor kinase 2 (GRK2) is localized to centrosomes and mediates epidermal growth factor-promoted centrosomal separation.

G protein-coupled receptor kinases (GRKs) play a central role in regulating receptor signaling, but recent studies suggest a broader role in modulating normal cellular functions. For example, GRK5 has been shown to localize to centrosomes and regulate microtubule nucleation and cell cycle progression. Here we demonstrate that GRK2 is also localized to centrosomes, although it has no role in centrosome duplication or microtubule nucleation. Of interest, knockdown of GRK2 inhibits epidermal growth factor receptor (EGFR)-mediated separation of duplicated centrosomes. This EGFR/GRK2-mediated process depends on the protein kinases mammalian STE20-like kinase 2 (Mst2) and Nek2A but does not involve polo-like kinase 1. In vitro analysis and dominant-negative approaches reveal that GRK2 directly phosphorylates and activates Mst2. Collectively these findings demonstrate that GRK2 is localized to centrosomes and plays a central role in mitogen-promoted centrosome separation most likely via its ability to phosphorylate Mst2.

G protein-coupled receptor kinase 5 phosphorylates nucleophosmin and regulates cell sensitivity to polo-like kinase 1 inhibition.

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, leading to their desensitization and endocytosis. GRKs have also been implicated in phosphorylating other classes of proteins and can localize in a variety of cellular compartments, including the nucleus. Here, we attempted to identify potential nuclear substrates for GRK5. Our studies reveal that GRK5 is able to interact with and phosphorylate nucleophosmin (NPM1) both in vitro and in intact cells. NPM1 is a nuclear protein that regulates a variety of cell functions including centrosomal duplication, cell cycle control, and apoptosis. GRK5 interaction with NPM1 is mediated by the N-terminal domain of each protein, and GRK5 primarily phosphorylates NPM1 at Ser-4, a site shared with polo-like kinase 1 (PLK1). NPM1 phosphorylation by GRK5 and PLK1 correlates with the sensitivity of cells to undergo apoptosis with cells having higher GRK5 levels being less sensitive and cells with lower GRK5 being more sensitive to PLK1 inhibitor-induced apoptosis. Taken together, our results demonstrate that GRK5 phosphorylates Ser-4 in nucleophosmin and regulates the sensitivity of cells to PLK1 inhibition.

G Protein-coupled receptor kinase 5 is localized to centrosomes and regulates cell cycle progression.

G protein-coupled receptor kinases (GRKs) are important regulators of G protein-coupled receptor function and mediate receptor desensitization, internalization, and signaling. While GRKs also interact with and/or phosphorylate many other proteins and modify their function, relatively little is known about the cellular localization of endogenous GRKs. Here we report that GRK5 co-localizes with γ-tubulin, centrin, and pericentrin in centrosomes. The centrosomal localization of GRK5 is observed predominantly at interphase and although its localization is not dependent on microtubules, it can mediate microtubule nucleation of centrosomes. Knockdown of GRK5 expression leads to G2/M arrest, characterized by a prolonged G2 phase, which can be rescued by expression of wild type but not catalytically inactive GRK5. This G2/M arrest appears to be due to increased expression of p53, reduced activity of aurora A kinase and a subsequent delay in the activation of polo-like kinase 1. Overall, these studies demonstrate that GRK5 is localized in the centrosome and regulates microtubule nucleation and normal cell cycle progression.

Differential expression of arrestins is a predictor of breast cancer progression and survival.

Emerging evidence has implicated G protein-coupled receptors, such as CXCR4 and PAR2, in breast cancer progression and the development of metastatic breast cancer. However, the role of proteins that regulate the function of these receptors, such as arrestins, in breast cancer has yet to be determined. Examination of the expression of the two nonvisual arrestins, arrestin2 and 3, in various breast cancer cell lines revealed comparable expression of arrestin3 in basal and luminal lines while arrestin2 expression was much higher in the luminal lines compared to the more aggressive basal lines. Analysis of normal human breast tissue revealed that arrestin2 and 3 were expressed in both luminal and myoepithelial cells of mammary epithelia with arrestin2 highest in myoepithelial cells and arrestin3 comparable in both cell types. Quantitative immunofluorescence-based examination of primary breast tumors revealed that arrestin2 expression significantly decreased with cancer progression from ductal carcinoma in situ to invasive carcinoma and further to lymph node metastasis (P < 0.001). Moreover, decreased arrestin2 expression was associated with decreased survival (P = 0.0007) as well as positive lymph node status and increased tumor size and nuclear grade. In contrast, arrestin3 expression significantly increased during breast cancer progression (P < 0.001) and increased expression was associated with decreased survival (P = 0.014). Arrestin3 was also an independent prognostic marker of breast cancer with a hazard ratio of 1.65. Overall, these studies demonstrate that arrestin2 levels decrease while arrestin3 levels increase during breast cancer progression and these changes correlate with a poor clinical outcome.

Non-visual arrestins are constitutively associated with the centrosome and regulate centrosome function.

In addition to regulating receptor activity, non-visual arrestins function as scaffolds for numerous intracellular signaling cascades and as regulators of gene transcription. Here we report that the two non-visual arrestins, arrestin2 and arrestin3, localize to the centrosome, a key organelle involved in microtubule nucleation and bipolar mitotic spindle assembly. Both arrestins co-localized with the centrosomal marker gamma-tubulin during interphase and mitosis and were found in purified centrosome preparations. In vitro binding assays demonstrated that both arrestins directly interact with gamma-tubulin. Knockdown of either arrestin by RNA interference resulted in multinucleation, centrosome amplification, and mitotic defects, although only the loss of arrestin2 triggered aberrant microtubule nucleation. Importantly, overexpression of wild type arrestin rescued the multinucleation phenotype and restored normal centrosome number in arrestin siRNA-transfected cells. Moreover, overexpression of arrestin2 or -3 rescued the multinucleation defect observed in MDA-MB-231 breast cancer cells. Taken together, our data reveal that non-visual arrestins are novel centrosomal components and regulate normal centrosome function.

Identification of betaArrestin2 as a corepressor of androgen receptor signaling in prostate cancer.

Androgen receptor (AR) signaling regulates the development and homeostasis of male reproductive organs, including the prostate. Deregulation of AR and AR coregulators, expression, or activity is involved in the initiation of prostate cancer and contributes to the transition of the disease to hormone-refractory stage. The ubiquitous betaArrestin proteins are now recognized as bona fide adapters and signal transducers with target effectors found in both the cytosol and nucleus. Here, we provide evidence that betaArrestin2 forms a complex with AR and acts as an AR corepressor in androgen-dependent prostate cancer cells. Accordingly, the forced overexpression of betaArrestin2 diminishes, and knockdown of betaArrestin2 expression with RNAi increases the androgen-induced prostate-specific antigen (PSA) gene expression. betaArrestin2 serves as an adapter, bringing into close proximity the Mdm2 E3 ligase and AR, thereby promoting AR ubiquitylation and degradation. Human prostate tissues evidence an inverse relationship between the expression of betaArrestin2 and AR activity: glands that express high levels of betaArrestin2 exhibit low expression of PSA, and those glands that express low levels of betaArrestin2 evidence elevated PSA levels. We conclude that betaArrestin2 acts as a corepressor of AR by serving as a scaffold for Mdm2 leading to the AR ubiquitylation and degradation.

STa peptide analogs for probing guanylyl cyclase C.

Guanylyl cyclase C (GC-C), universally overexpressed on primary and metastatic colorectal carcinoma cells, is activated by endogenous ligands, guanylin, and uroguanylin, and by exogenous 18-residue heat-stable enterotoxins (STa) produced by diarrheagenic bacteria. Two 12-residue STa analogs with alternate combinations of two interlocked disulfide bonds, peptides 3 and 6, were synthesized by orthogonal solid phase synthesis routes. Peptides 3 and 6 bound GC-C with a rank order potency of STa > peptide 3 > peptide 6. Peptides 3 and 6 behaved as agonists in stimulating cGMP production. The results reveal that the toxic domain of STa can be reduced to 12 amino acids.