Ion transporters: A new Cu-lprit for tyrosinase activation in melanosomes

The copper transporter ATP7A is required for tyrosinase activity and melanin production in melanosomes.

Melanosomes are specialized lysosome-related organelles where melanin precursors are formed from tyrosine in a reaction catalyzed by tyrosinase. Copper, an essential tyrosinase cofactor, is delivered to the trans-Golgi network in most cells by the transmembrane ion transporter ATP7A. However, the mechanism that permits copper accumulation and consequent tyrosinase activity in melanosomes remains poorly understood. In Nature, Setty et al. now report that an eight-subunit complex called BLOC-1 (biogenesis of lysosome-related organelles complex 1) regulates ATP7A trafficking to melanosomes and provides stringent spatial control over tyrosinase activation and melanin production.

Mutations in BLOC-1 subunits cause mislocalization of several melanosomal proteins and impair melanosome biogenesis. Immunofluorescence microscopy revealed that ATP7A, but not the related protein ATP7B, was present in secretory pathway components and mature melanosomes of normal melanocytes. However, in BLOC-1-deficient cells ATP7A was excluded from melanosomes and instead accumulated in perinuclear Golgi structures and endosomes. Reconstitution of an intact BLOC-1 complex restored the melanosomal localization of ATP7A and normal pigmentation, indicating that proper localization of ATP7A is dependent on BLOC-1. The authors' previous work had shown that tyrosinase localization is largely independent of BLOC-1.

Interestingly, despite normal tyrosinase expression and localization, tyrosinase activity in BLOC-1-deficient cells was restricted to the trans-Golgi network and melanin was not produced. Could this phenotype be attributed to defective ATP7A localization and hence a lack of melanosomal copper? Indeed, addition of copper restored tyrosinase activity in BLOC-1-deficient cells, resulting in increased pigmentation and melanin deposition in fixed melanocytes in vitro. Thus, both ATP7A localization and copper transport in melanosomes correlate with tyrosinase activity and normal melanin production.

A lingering question resulting from this work is why components of the trans-Golgi network are not pigmented, despite the common localization of tyrosinase and ATP7A in the secretory pathway. The authors hypothesize that copper binds inefficiently to tyrosinase in the trans-Golgi network and is lost during transport to melanosomes, in part because the acidic pH in secretory vesicles disrupts the copper-tyrosinase interaction. Once tyrosinase reaches the melanosome — which has a more alkaline pH — copper must be added anew, thus elucidating the observed requirement for functional ATP7A in this organelle. As the melanin precursors are toxic, tight spatial control of tyrosinase activation ensures that melanin is produced only within mature melanosomes. The external cues that regulate tyrosinase copper binding and destabilization remain to be fully explored.

Emily J. Chenette
Signaling Gateway

Original Reference:
Setty, S. R. G., Tenza, D., Sviderskaya, E. V., Bennett, D. C., Raposo, G. & Marks, M. S.
Cell-specific ATP7A transport sustains copper-dependent tyrosinase activity in melanosomes
Nature 454, 1142-1146 (2008)
Full text | PDF | Subscribe to Nature

Cell cycle: Coming together to tear cells apart

The sequential phosphorylation of PKCε is necessary for its association with 14-3-3 proteins and its localization to the mitotic cleavage furrow, as well as the subsequent dissolution of the mitotic ring and the separation of daughter cells during mitosis.

Daughter cells are separated in the final steps of mitosis through ingression of the cleavage furrow, which is driven by the constriction of an actomyosin ring. The regulated disassembly of the ring facilitates the physical separation of daughter cells (abscission). In Nature Cell Biology, Saurin et al. report that protein kinase C-ε (PKCε) activation and subsequent 14-3-3 binding regulates actomyosin ring dissolution.

A yeast two-hybrid screen revealed a specific interaction between PKCε and 14-3-3 proteins. In vitro analyses showed that the interaction occurred with multiple members of the 14-3-3 protein family and was regulated by phosphorylation of three conserved PKCε residues — Ser 368, Ser 350 and Ser 346. Phosphorylation of Ser 368 was found to be dependent on PKC, whereas p38 mitogen-activated protein kinase (MAPK)-mediated phosphorylation of Ser 350 generated a recognition site for glycogen synthase kinase 3 (GSK3) at Ser 346. The authors also found that phosphorylation of Ser 368 and Ser 346 was required for 14-3-3 association. These data suggest that the binding of PKCε to 14-3-3 is regulated by a stepwise phosphorylation cascade.

Immunofluorescence using phospho-specific antibodies demonstrated the specific activation of PKCε in mitotic cells and its accumulation at the cleavage furrow. Both knockdown of PKCε and expression of PKCε mutants unable to associate with 14-3-3 led to either delay or failure of cytokinesis, suggesting that both PKCε activity and 14-3-3 association are important for this process. A screen of proteins known to associate with the cleavage furrow during cytokinesis revealed that PKCε activity is necessary for the inactivation and relocalization of RhoA. Active, GTP-bound forms of RhoA accumulated at the furrow in the absence of functional PKCε, preventing dissociation of the actomyosin ring and abscission.

The presented data contribute to a model of abscission in which multiple cell signaling cascades converge upon PKCε, priming it for interaction with 14-3-3. Activated PKCε then localizes to the cleavage furrow and inhibits RhoA, leading to the dissolution of the actomyosin ring and abscission. However, the upstream signaling events are yet to be determined, as well as the mechanisms of temporal and spatial regulation of RhoA activity by PKCε at the cleavage furrow.

Anna S. Kushnir
Nature Publishing Group

Original Reference:
Saurin, A. et al.
The regulated assembly of a PKCε complex controls the completion of cytokinesis
Nature Cell Biology 10, 891-901 (2008)
Full text | PDF | Subscribe to Nature Cell Biology

Polycystic kidney disease: The TNF-α connection

TNF-α-mediated induction of FIP2 causes mislocalization of polycystin-2 and potentiates the development of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the polycystin-1 (PKD1) or polycystin-2 (PKD2) genes. However, a mouse model of the disease has shown that a reduction in Pkd1 expression fosters the development of cysts, implicating other, non-genetic factors in ADPKD progression. In Nature Medicine, Li et al. report that tumor necrosis factor-α (TNF-α)-mediated induction of the scaffold protein FIP2 (also known as optineurin) potentiates cyst formation in Pkd2^+/- mice, and that pharmacologic inhibition of TNF-α blocks disease progression.

TNF-α is upregulated following renal injury, but a connection between TNF-α and ADPKD had not yet been documented. Li et al. found that TNF-α treatment caused a redistribution of polycystin-2 from the plasma membrane and cilia to perinuclear structures. Polycystin-2 bound to FIP2 — a TNF-α-inducible protein with a known role in Rab8a-mediated trafficking — and FIP2 knockdown restored normal polycystin-2 localization. TNF-α treatment also disrupted the functional interaction between polycystin-1 and polycystin-2, although it did not affect polycystin-1 localization.

TNF-α was detected in the cyst fluid of ADPKD patients, and FIP2 protein levels in the cyst-lining cells of these patients were twice as high as those in normal kidney cells. Furthermore, Pkd2^+/- embryonic mouse kidneys formed cysts ex vivo and expressed high levels of FIP2 and TNF-α receptor 1 (TNFR-1); TNF-α treatment further upregulated FIP2 expression and potentiated cyst formation both ex vivo and in vivo. Intriguingly, administration of the TNF-α inhibitor etanercept blocked kidney cyst formation in Pkd2^+/- mice irrespective of whether they had been treated with exogenous TNF-α.

These data suggest that TNF-α-induced FIP2 promotes the development of ADPKD by sequestering polycystin-2 and disrupting the functional polycystin-1/polycystin-2 complex. TNF-α expression may promote or aggravate the development of ADPKD in individuals with a somatic PKD2 mutation, thus alleviating the need for a 'second hit' at the PKD1/2 locus and raising the possibility that TNF-α inhibitors could be effective in treating ADPKD. As elevated TNF-α levels have been detected in the cystic kidneys of a mouse model of autosomal recessive polycystic kidney disease (ARPKD), it will be interesting to determine if inhibition of TNF-α signaling affects the progression of other cystic kidney diseases.

Emily J. Chenette
Signaling Gateway

Original Reference:
Li, X. et al.
A tumor necrosis factor-α-mediated pathway promoting autosomal dominant polycystic kidney disease
Nature Medicine 14, 863-868 (2008)
Full text | PDF | Subscribe to Nature Medicine

PI(3)K signaling: Separate but not equal

The kinase activity of the PI(3)K subunit p110β is linked to the oncogenic transformation of mouse prostate epithelium, whereas a second, kinase-independent activity regulates cellular proliferation and signaling downstream of tyrosine-kinase receptors.

Class IA phosphatidylinositol-3-OH kinases (PI(3)Ks) are heterodimeric lipid and protein kinases composed of a regulatory subunit (p50α, p55α, p85α, p55γ or p85β) and a catalytic subunit (p110α, p110β or p110δ). PI(3)Ks regulate cell growth and proliferation, and the separable biological activities of the different catalytic subunits are under intense investigation. The ability of p110α to promote angiogenesis and tumor development is well established, but information about the physiological role of p110β is lacking. In Nature, Jean Zhao, Thomas Roberts and colleagues now report that p110β-associated kinase activity is required for the oncogenic transformation of mouse prostate epithelium, whereas a second, kinase-independent activity regulates cell proliferation and signaling downstream of tyrosine–kinase receptors.

p110β has been shown to phosphorylate Akt downstream of the lysophosphatidic acid (LPA) G protein-coupled receptor (GPCR), but not the insulin tyrosine–kinase receptor. In keeping with these observations, p110β-deficient mouse embryonic fibroblasts (MEFs) proliferated slowly and did not activate Akt or its downstream target S6 ribosomal protein (S6RP) in response to LPA. As expected, insulin treatment had no effect on Akt phosphorylation in p110β-deficient MEFs; however, S6RP phosphorylation was moderately depressed. Interestingly, expression of a kinase-deficient p110β^K805R mutant was sufficient to restore S6RP phosphorylation in response to insulin, whereas exogenous wild-type p110β was required for Akt/S6RP phosphorylation in response to LPA. In addition, p110β^K805R expression permitted normal cell cycle progression in p110β-deficient MEFs, suggesting a kinase-independent role of p110β in cellular proliferation and signaling downstream of the insulin receptor. The nature of the kinase-independent activity is not yet known, nor has a kinase-dependent role for p110β been ruled out in these processes.

Previous studies have indicated that p110β lipid kinase activity is required for prostate cancer progression, and Zhao and colleagues confirmed that loss of p110β blocked tumorigenesis in PTEN-deficient anterior prostatic epithelium. Surprisingly, p110α depletion did not affect either tumor development or Akt phosphorylation. These findings suggest that p110β catalytic activity drives transformation and Akt phosphorylation in the mouse prostate epithelium in the absence of PTEN.

Thus, distinct biological functions for p110α and p110β are beginning to emerge. In a previous report in Nature, Holger Gerhardt, Bart Vanhaesebroeck and colleagues described p110α as a key factor in angiogenesis and endothelial cell migration, whereas Zhao and Roberts' study found a kinase-independent function for p110β in aspects of growth-factor signaling and cell proliferation, and a kinase-dependent role in oncogenic transformation. Even though p110α remains the focus of pharmaceutical efforts to block oncogenic PI(3)K signaling, these findings suggest that p110β may be an equally attractive drug target.

Emily J. Chenette
Signaling Gateway

Original Reference:
Jia, S., Liu, Z., Zhang, S., Liu, P., Zhang, L., Lee, S. H., Zhang, J., Signoretti, S., Loda, M., Roberts, T. M. & Zhao, J. J.
Essential roles of PI(3)K–p110β in cell growth, metabolism and tumorigenesis
Nature 454, 776-779 (2008)
Full text | PDF | Subscribe to Nature

Graupera, M., Guillermet-Guibert, J., Foukas, L. C., Phng, L.-K. Cain, R. J., Salpekar, A., Pearce, W., Meek, S., Millan, J., Cutillas, P. R., Smith, A. J. H., Ridley, A. J., Ruhrberg, C., Gerhardt H. & Vanhaesebroeck B.
Angiogenesis selectively requires the p110α isoform of PI3K to control endothelial cell migration
Nature 454, 662-666 (2008)
Full text | PDF | Subscribe to Nature

Immunology: Arrestin cytotoxic pathway activation

β-arrestin 2 negatively regulates natural killer (NK) cell cytotoxicity by facilitating recruitment of Src homology-containing tyrosine phosphatases (SHPs) to inhibitory NK cell receptors.

Natural killer (NK) cells are an important part of the innate immune response because they recognize and kill virus-infected or cancerous cells. NK cell activity is regulated by the opposing actions of activating killer immunoglobulin-like receptors (KIRs), which sense infected cells and stimulate cytotoxic signaling, and inhibitory KIRs, which recognize healthy cells. After stimulation, phosphorylated inhibitory KIRs recruit Src homology-containing tyrosine phosphatases (SHPs) that then dephosphorylate and inactivate cytotoxic signaling molecules such as Vav and Erk. In Nature Immunology, Yu et al. now report that the scaffold protein β-arrestin 2 associates with phosphorylated KIRs, which facilitates SHP recruitment and downregulates the cytotoxic activity of NK cells.

A yeast two-hybrid screen for regulators of inhibitory KIR signaling identified β-arrestin 2 as specifically interacting with the phosphorylated membrane-proximal immunoreceptor tyrosine-based inhibitor motif (ITIM) of the endogenous KIR2DL1 inhibitory receptor. Immunoprecipitation assays revealed that KIR2DL1, β-arrestin 2 and either SHP-1 or SHP-2 formed a ternary complex. Although a direct interaction between β-arrestin 2 and SHP-1 or SHP-2 was not detected, overexpression of β-arrestin 2 increased the amount of SHP-1 or SHP-2 recruited to KIR2DL1. siRNA against β-arrestin 2, however, reduced this association.

Stimulation of KIR2DL1 causes SHP-mediated dephosphorylation of Erk and Vav. Depletion of β-arrestin 2 increased Erk activation and promoted NK cell-directed lysis in response to a normally inhibitory ligand. β-arrestin 2 knockdown also increased the accumulation of granzyme B at the junction of NK cells and their targets — a necessary prerequisite for NK-mediated cell lysis. Mice overexpressing β-arrestin 2 had higher mouse cytomegalovirus (MCMV) viral titers than their wild-type counterparts, and NK cells from these mice were less efficient in killing lymphoma cells or tumor cells in vivo. Conversely, β-arrestin 2-deficient NK cells displayed higher cytotoxicity towards target cells.

These data reveal a novel role for β-arrestin 2 in regulating inhibitory KIR signaling. Although the mechanistic details of SHP binding to phosphorylated ITIMs and β-arrestin 2 remain to be elucidated, β-arrestin 2 has been shown to serve as a scaffold for the Raf–MEK1–Erk pathway, suggesting that β-arrestin 2 may recruit SHP substrates following inhibitory KIR activation. Furthermore, β-arrestins regulate the internalization of some G protein-coupled receptors (GPCRs). It will be interesting to determine whether β-arrestins can promote GPCR dephosphorylation by recruiting SHPs in other contexts.

Emily J. Chenette
Signaling Gateway

Original Reference:
Yu, M.-C. et al.
An essential function for β-arrestin 2 in the inhibitory signaling of natural killer cells
Nature Immunology 9, 898-907 (2008)
Full text | PDF | Subscribe to Nature Immunology

Bryceson, Y. T. & Ljunggren, H.-G.
Arrestin NK cell cytotoxicity
Nature Immunology 9, 835-836 (2008)
Full text | PDF | Subscribe to Nature Immunology