Structure of human nSMase2 reveals an interdomain allosteric activation mechanism for ceramide generation.

Neutral sphingomyelinase 2 (nSMase2, product of the SMPD3 gene) is a key enzyme for ceramide generation that is involved in regulating cellular stress responses and exosome-mediated intercellular communication. nSMase2 is activated by diverse stimuli, including the anionic phospholipid phosphatidylserine. Phosphatidylserine binds to an integral-membrane N-terminal domain (NTD); however, how the NTD activates the C-terminal catalytic domain is unclear. Here, we identify the complete catalytic domain of nSMase2, which was misannotated because of a large insertion. We find the soluble catalytic domain interacts directly with the membrane-associated NTD, which serves as both a membrane anchor and an allosteric activator. The juxtamembrane region, which links the NTD and the catalytic domain, is necessary and sufficient for activation. Furthermore, we provide a mechanistic basis for this phenomenon using the crystal structure of the human nSMase2 catalytic domain determined at 1.85-Å resolution. The structure reveals a DNase-I-type fold with a hydrophobic track leading to the active site that is blocked by an evolutionarily conserved motif which we term the "DK switch." Structural analysis of nSMase2 and the extended N-SMase family shows that the DK switch can adopt different conformations to reposition a universally conserved Asp (D) residue involved in catalysis. Mutation of this Asp residue in nSMase2 disrupts catalysis, allosteric activation, stimulation by phosphatidylserine, and pharmacological inhibition by the lipid-competitive inhibitor GW4869. Taken together, these results demonstrate that the DK switch regulates ceramide generation by nSMase2 and is governed by an allosteric interdomain interaction at the membrane interface.

Glycoprotein A repetitions predominant (GARP) positively regulates transforming growth factor (TGF) ß3 and is essential for mouse palatogenesis.

Glycoprotein A repetitions predominant (GARP) (encoded by the Lrrc32 gene) plays important roles in cell-surface docking and activation of TGFß. However, GARP's role in organ development in mammalian systems is unclear. To determine the function of GARP in vivo, we generated a GARP KO mouse model. Unexpectedly, the GARP KO mice died within 24 h after birth and exhibited defective palatogenesis without apparent abnormalities in other major organs. Furthermore, we observed decreased apoptosis and SMAD2 phosphorylation in the medial edge epithelial cells of the palatal shelf of GARP KO embryos at embryonic day 14.5 (E14.5), indicating a defect in the TGFß signaling pathway in the GARP-null developing palates. Of note, the failure to develop the secondary palate and concurrent reduction of SMAD phosphorylation without other defects in GARP KO mice phenocopied TGFß3 KO mice, although GARP has not been suggested previously to interact with TGFß3. We found that GARP and TGFß3 co-localize in medial edge epithelial cells at E14.5. In vitro studies confirmed that GARP and TGFß3 directly interact and that GARP is indispensable for the surface expression of membrane-associated latent TGFß3. Our findings indicate that GARP is essential for normal morphogenesis of the palate and demonstrate that GARP plays a crucial role in regulating TGFß3 signaling during embryogenesis. In conclusion, we have uncovered a novel function of GARP in positively regulating TGFß3 activation and function.

Role of neutral ceramidase in colon cancer.

Alterations in sphingolipid metabolism, especially ceramide and sphingosine 1-phosphate, have been linked to colon cancer, suggesting that enzymes of sphingolipid metabolism may emerge as novel regulators and targets in colon cancer. Neutral ceramidase (nCDase), a key enzyme in sphingolipid metabolism that hydrolyzes ceramide into sphingosine, is highly expressed in the intestine; however, its role in colon cancer has not been defined. Here we show that molecular and pharmacological inhibition of nCDase in colon cancer cells increases ceramide, and this is accompanied by decreased cell survival and increased apoptosis and autophagy, with minimal effects on noncancerous cells. Inhibition of nCDase resulted in loss of ß-catenin and inhibition of ERK, components of pathways relevant for colon cancer development. Furthermore, inhibition of nCDase in a xenograft model delayed tumor growth and increased ceramide while decreasing proliferation. It is noteworthy that mice lacking nCDase treated with azoxymethane were protected from tumor formation. Taken together, these studies show that nCDase is pivotal for regulating initiation and development of colon cancer, and these data suggest that this enzyme is a suitable and novel target for colon cancer therapy.-García-Barros, M., Coant, N., Kawamori, T., Wada, M., Snider, A. J., Truman, J.-P., Wu, B. X., Furuya, H., Clarke, C. J., Bialkowska, A. B., Ghaleb, A., Yang, V. W., Obeid, L. M., Hannun, Y. A. Role of neutral ceramidase in colon cancer.

Complexity of Chronic Conditions' Impact on End-of-Life Expense Trajectories of Medicare Decedents.

OBJECTIVE: The aim of this study is to determine if the pattern of monthly medical expense can be used to identify individuals at risk of dying, thus supporting providers in proactively engaging in advanced care planning discussions. BACKGROUND: Identifying the right time to discuss end of life can be difficult. Improved predictive capacity has made it possible for nurse leaders to use large data sets to guide clinical decision making. METHODS: We examined the patterns of monthly medical expense of Medicare beneficiaries with life-limiting illness during the last 24 months of life using analysis of variance, t tests, and stepwise hierarchical linear modeling. RESULTS: In the final year of life, monthly medical expense increases rapidly for all disease groupings and forms distinct patterns of change. CONCLUSION: Type of condition can be used to classify decedents into distinctly different cost trajectories. Conditions including chronic disease, system failure, or cancer may be used to identify patients who may benefit from supportive care.

Essential roles of grp94 in gut homeostasis via chaperoning canonical Wnt pathway.

Increasing evidence points to a role for the protein quality control in the endoplasmic reticulum (ER) in maintaining intestinal homeostasis. However, the specific role for general ER chaperones in this process remains unknown. Herein, we report that a major ER heat shock protein grp94 interacts with MesD, a critical chaperone for the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6). Without grp94, LRP6 fails to export from the ER to the cell surface, resulting in a profound loss of canonical Wnt signaling. The significance of this finding is demonstrated in vivo in that grp94 loss causes a rapid and profound compromise in intestinal homeostasis with gut-intrinsic defect in the proliferation of intestinal crypts, compromise of nuclear ß-catenin translocation, loss of crypt-villus structure, and impaired barrier function. Taken together, our work has uncovered the role of grp94 in chaperoning LRP6-MesD in coordinating intestinal homeostasis, placing canonical Wnt-signaling pathway under the direct regulation of the general protein quality control machinery in the ER.

Patient Readiness to Exercise After Cardiac Surgery: Development of the Readiness to Change Exercise Questionnaire.

BACKGROUND: Readiness to change plays a significant role in patient adherence to an exercise regimen; thus, accurate assessment of readiness to change is necessary to direct interventions. To date, an accurate scale for measuring readiness to exercise after cardiac surgery is not available. OBJECTIVES: The purpose of this study was to develop the Readiness to Change Exercise Questionnaire for use among Thai cardiac surgery patients and to evaluate its psychometric properties. METHODS: The Readiness to Change Exercise Questionnaire was developed based on the Transtheoretical Model, a comprehensive literature review, and input from experts and cardiac surgery patients. Participants were 533 patients who had undergone cardiac surgery within the previous 3 months. The study was conducted in 7 hospitals in 4 geographical regions of Thailand. RESULTS: Confirmatory factor analysis showed satisfactory goodness of fit for the 13-item scale. The analysis supported a 4-factor structure corresponding to 4 readiness stages: precontemplation, contemplation, preparation, and action. Cronbach's α coefficients were .68 for precontemplation, .75 for contemplation, .72 for preparation, and .75 for action. CONCLUSIONS: The scale was found to be a valid and reliable instrument for the determination of patient readiness to exercise after cardiac surgery. However, further testing of the scale is needed to confirm its concurrent and predictive validity.

Defining a role for acid sphingomyelinase in the p38/interleukin-6 pathway.

Acid sphingomyelinase (ASM) is one of the key enzymes involved in regulating the metabolism of the bioactive sphingolipid ceramide in the sphingolipid salvage pathway, yet defining signaling pathways by which ASM exerts its effects has proven difficult. Previous literature has implicated sphingolipids in the regulation of cytokines such as interleukin-6 (IL-6), but the specific sphingolipid pathways and mechanisms involved in inflammatory signaling need to be further elucidated. In this work, we sought to define the role of ASM in IL-6 production because our previous work showed that a parallel pathway of ceramide metabolism, acid ß-glucosidase 1, negatively regulates IL-6. First, silencing ASM with siRNA abrogated IL-6 production in response to the tumor promoter, 4ß-phorbol 12-myristate 13-acetate (PMA), in MCF-7 cells, in distinction to acid ß-glucosidase 1 and acid ceramidase, suggesting specialization of the pathways. Moreover, treating cells with siRNA to ASM or with the indirect pharmacologic inhibitor desipramine resulted in significant inhibition of TNFα- and PMA-induced IL-6 production in MDA-MB-231 and HeLa cells. Knockdown of ASM was found to significantly inhibit PMA-dependent IL-6 induction at the mRNA level, probably ruling out mechanisms of translation or secretion of IL-6. Further, ASM knockdown or desipramine blunted p38 MAPK activation in response to TNFα, revealing a key role for ASM in activating p38, a signaling pathway known to regulate IL-6 induction. Last, knockdown of ASM dramatically blunted invasion of HeLa and MDA-MB-231 cells through Matrigel. Taken together, these results demonstrate that ASM plays a critical role in p38 signaling and IL-6 synthesis with implications for tumor pathobiology.

Endoplasmic reticulum heat shock protein gp96 maintains liver homeostasis and promotes hepatocellular carcinogenesis.

BACKGROUND & AIMS: gp96, or grp94, is an endoplasmic reticulum (ER)-localized heat shock protein 90 paralog that acts as a protein chaperone and plays an important role for example in ER homeostasis, ER stress, Wnt and integrin signaling, and calcium homeostasis, which are vital processes in oncogenesis. However, the cancer-intrinsic function of gp96 remains controversial. METHODS: We studied the roles of gp96 in liver biology in mice via an Albumin promoter-driven Cre recombinase-mediated disruption of gp96 gene, hsp90b1. The impact of gp96 status on hepatic carcinogenesis in response to diethyl-nitrosoamine (DENA) was probed. The roles of gp96 on human hepatocellular carcinoma cells (HCC) were also examined pharmacologically with a targeted gp96 inhibitor. RESULTS: We demonstrated that gp96 maintains liver development and hepatocyte function in vivo, and its loss genetically promotes adaptive accumulation of long chain ceramides, accompanied by steatotic regeneration of residual gp96+ hepatocytes. The need for compensatory expansion of gp96+ cells in the gp96- background predisposes mice to develop carcinogen-induced hepatic hyperplasia and cancer from gp96+ but not gp96- hepatocytes. We also found that genetic and pharmacological inhibition of gp96 in human HCCs perturbed multiple growth signals, and attenuated proliferation and expansion. CONCLUSIONS: gp96 is a pro-oncogenic chaperone and an attractive therapeutic target for HCC.

Targeting (cellular) lysosomal acid ceramidase by B13: design, synthesis and evaluation of novel DMG-B13 ester prodrugs.

Acid ceramidase (ACDase) is being recognized as a therapeutic target for cancer. B13 represents a moderate inhibitor of ACDase. The present study concentrates on the lysosomal targeting of B13 via its N,N-dimethylglycine (DMG) esters (DMG-B13 prodrugs). Novel analogs, the isomeric mono-DMG-B13, LCL522 (3-O-DMG-B13·HCl) and LCL596 (1-O-DMG-B13·HCl) and di-DMG-B13, LCL521 (1,3-O, O-DMG-B13·2HCl) conjugates, were designed and synthesized through N,N-dimethyl glycine (DMG) esterification of the hydroxyl groups of B13. In MCF7 cells, DMG-B13 prodrugs were efficiently metabolized to B13. The early inhibitory effect of DMG-B13 prodrugs on cellular ceramidases was ACDase specific by their lysosomal targeting. The corresponding dramatic decrease of cellular Sph (80-97% Control/1h) by DMG-B13 prodrugs was mainly from the inhibition of the lysosomal ACDase.

hkb is required for DIP-α expression and target recognition in the Drosophila neuromuscular circuit.

Our nervous system contains billions of neurons that form precise connections with each other through interactions between cell surface proteins. In Drosophila, the Dpr and DIP immunoglobulin protein subfamilies form homophilic or heterophilic interactions to instruct synaptic connectivity, synaptic growth, and cell survival. However, the upstream regulatory mechanisms of Dprs and DIPs are not clear. On the other hand, while transcription factors have been implicated in target recognition, their downstream cell surface proteins remain mostly unknown. We conduct an F1 dominant modifier genetic screen to identify regulators of Dprs and DIPs. We identify huckebein (hkb), a transcription factor previously implicated in target recognition of the dorsal Is motor neuron. We show that hkb genetically interacts with DIP-α and loss of hkb leads to complete removal of DIP-α expression specifically in dorsal Is motor neurons. We then confirm that this specificity is through the dorsal Is motor neuron specific transcription factor, even-skipped (eve), which acts downstream of hkb. Analysis of the genetic interaction between hkb and eve reveals that they act in the same pathway to regulate dorsal Is motor neuron connectivity. Our study provides insight into the transcriptional regulation of DIP-α and suggests that distinct regulatory mechanisms exist for the same CSP in different neurons.

Evaluation of the role of secretory sphingomyelinase and bioactive sphingolipids as biomarkers in hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a rare systemic inflammatory syndrome that results from unrestrained immune cell activation. Despite significant advances in the understanding of the pathophysiology of HLH, interventions remain limited for this often-fatal condition. Secretory sphingomyelinase (S-SMase) is a pro-inflammatory lipid hydrolase that is upregulated in several inflammatory conditions, including HLH. S-SMase promotes the formation of ceramide, a bioactive lipid implicated in several human disease states. However, the role of the S-SMase/ceramide pathway in HLH remains unexplored. To further evaluate the role of S-SMase upregulation in HLH, we tested the serum of patients with HLH (n = 16; primary = 3, secondary = 13) and healthy control patients (n = 25) for serum S-SMase activity with tandem sphingolipid metabolomic profiling. Patients with HLH exhibited elevated levels of serum S-SMase activity, with concomitant elevations in several ceramide species and sphingosine, while levels of sphingosine-1-phosphate were significantly decreased. Importantly, the ratio of C16 -ceramide:sphingosine was uniquely elevated in HLH patients that died despite appropriate treatment, but remained low in HLH patients that survived, suggesting that this ratio may be of prognostic significance. Together, these results demonstrate upregulation of the S-SMase/ceramide pathway in HLH, and suggest that the balance of ceramide and sphingosine determine clinical outcomes in HLH. .

Second language listening difficulties perceived by low-level learners.

The purpose was the develop a questionnaire to identify the specific listening difficulties of second language (L2) learners. Based on previous research, a questionnaire containing 31 items was developed and administered to 1,056 college freshmen. The whole sample was split randomly into two subsamples, each containing 528 cases. Exploratory factor analysis was performed to analyse the first subsample, and six factors were extracted, explaining a total of 57.1% of variance. To test the factor model, confirmatory factor analysis was conducted with the second subsample. Various fit indices were examined. The best fitting model for the data was a 23-item, six-factor model representing text, input channel and surroundings, relevance, listener, speaker, and task. Apart from the listener factor, all components are external ones and deemed to be uncontrollable by listeners. L2 learners must take an active role in listening practice to overcome L2 listening difficulties.

hkb is required for DIP-α expression and target recognition in the Drosophila neuromuscular circuit.

Our nervous system contains billions of neurons that form precise connections with each other through interactions between cell surface proteins (CSPs). In Drosophila, the Dpr and DIP immunoglobulin protein subfamilies form homophilic or heterophilic interactions to instruct synaptic connectivity, synaptic growth and cell survival. However, the upstream regulation and downstream signaling mechanisms of Dprs and DIPs are not clear. In the Drosophila larval neuromuscular system, DIP-α is expressed in the dorsal and ventral type-Is motor neurons (MNs). We conducted an F1 dominant modifier genetic screen to identify regulators of Dprs and DIPs. We found that the transcription factor, huckebein (hkb), genetically interacts with DIP-α and is important for target recognition specifically in the dorsal Is MN, but not the ventral Is MN. Loss of hkb led to complete removal of DIP-α expression. We then confirmed that this specificity is through the dorsal Is MN specific transcription factor, even-skipped (eve), which acts downstream of hkb. Genetic interaction between hkb and eve revealed that they act in the same pathway to regulate dorsal Is MN connectivity. Our study provides insight into the transcriptional regulation of DIP-α and suggests that distinct regulatory mechanisms exist for the same CSP in different neurons.

Identification of novel anionic phospholipid binding domains in neutral sphingomyelinase 2 with selective binding preference.

Sphingolipids such as ceramide are recognized as vital regulators of many biological processes. Neutral sphingomyelinase 2 (nSMase2) is one of the key enzymes regulating ceramide production. It was previously shown that the enzymatic activity of nSMase2 was dependent on anionic phospholipids (APLs). In this study, the structural requirements for APL-selective binding of nSMase2 were determined and characterized. Using lipid-protein overlay assays, nSMase2 interacted specifically and directly with several APLs, including phosphatidylserine and phosphatidic acid. Lipid-protein binding studies of deletion mutants identified two discrete APL binding domains in the N terminus of nSMase2. Further, mutagenesis experiments pinpointed the core sequences and major cationic amino acids in the domains that are necessary for the cooperative activation of nSMase2 by APLs. The first domain included the first amino-terminal hydrophobic segment and Arg-33, which were essential for nSMase2 to interact with APLs. The second binding domain was comprised of the second hydrophobic segment and Arg-92 and Arg-93. Moreover, mutation of one or both domains decreased APL binding and APL-dependent catalytic activity of nSMase2. Further, mutation of both domains in nSMase2 reduced its plasma membrane localization. Finally, these binding domains are also important for the capability of nSMase2 to rescue the defects of yeast lacking the nSMase homologue, ISC1. In conclusion, these data have identified the APL binding domains of nSMase2 for the first time. The analysis of interactions between nSMase2 and APLs will contribute to our understanding of signaling pathways mediated by sphingolipid metabolites.

Novel pathway of ceramide production in mitochondria: thioesterase and neutral ceramidase produce ceramide from sphingosine and acyl-CoA.

Reports suggest that excessive ceramide accumulation in mitochondria is required to initiate the intrinsic apoptotic pathway and subsequent cell death, but how ceramide accumulates is unclear. Here we report that liver mitochondria exhibit ceramide formation from sphingosine and palmitoyl-CoA and from sphingosine and palmitate. Importantly, this activity was markedly decreased in liver from neutral ceramidase (NCDase)-deficient mice. Moreover, the levels of ceramide were dissimilar in liver mitochondria of WT and NCDase KO mice. These results suggest that NCDase is a key participant of ceramide formation in liver mitochondria. We also report that highly purified liver mitochondria have ceramidase, reverse ceramidase, and thioesterase activities. Increased accessibility of palmitoyl-CoA to the mitochondrial matrix with the pore-forming peptide zervamicin IIB resulted in 2-fold increases in palmitoyl-CoA hydrolysis by thioesterase. This increased hydrolysis was accompanied by an increase in ceramide formation, demonstrating that both outer membrane and matrix localized thioesterases can regulate ceramide formation. Also, ceramide formation might occur both in the outer mitochondrial membrane and in the mitochondrial matrix, suggesting the existence of distinct ceramide pools. Taken together, these results suggest that the reverse activity of NCDase contributes to sphingolipid homeostasis in this organelle in vivo.

Regulation of CC ligand 5/RANTES by acid sphingomyelinase and acid ceramidase.

Acid sphingomyelinase (aSMase) generates the bioactive lipid ceramide (Cer) from hydrolysis of sphingomyelin (SM). However, its precise roles in regulating specific sphingolipid-mediated biological processes remain ill defined. Interestingly, the aSMase gene gives rise to two distinct enzymes, lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase) via alternative trafficking of a shared protein precursor. Previously, our laboratory identified Ser(508) as a crucial residue for the constitutive and regulated secretion of S-SMase in response to inflammatory cytokines, and demonstrated a role for S-SMase in formation of select cellular Cer species (Jenkins, R. W., Canals, D., Idkowiak-Baldys, J., Simbari, F., Roddy, P., Perry, D. M., Kitatani, K., Luberto, C., and Hannun, Y. A. (2010) J. Biol. Chem. 285, 35706-35718). In the present study using a chemokine/cytokine screen, we identified the chemokine CCL5 (formerly known as RANTES) as a candidate-specific downstream target for aSMase. Regulation of CCL5 by aSMase was subsequently validated using both loss-of-function and gain-of-function models indicating that aSMase is both necessary and sufficient for CCL5 production. Interestingly, cells deficient in acid ceramidase (aCDase) also exhibited defects in CCL5 induction, whereas cells deficient in sphingosine kinase-1 and -2 exhibited higher levels of CCL5, suggesting that sphingosine and not sphingosine 1-phosphate (S1P) is responsible for the positive signal to CCL5. Consistent with this, co-expression of aSMase and aCDase was sufficient to strongly induce CCL5. Taken together, these data identify a novel role for aSMase (particularly S-SMase) in chemokine elaboration by pro-inflammatory cytokines and highlight a novel and shared function for aSMase and aCDase.

Loss of neutral ceramidase increases inflammation in a mouse model of inflammatory bowel disease.

Sphingolipids are emerging as important mediators of immune and inflammatory responses. We have previously demonstrated that sphingosine-1-phosphate (S1P) and its synthetic enzyme sphingosine kinase-1 (SK1) play an important role in inflammatory bowel disease. S1P generation is dependent on SK phosphorylation of sphingosine. Generation of sphingosine results only from the breakdown of ceramide by ceramidases (CDase). In this study, we set out to determine the role of neutral CDase (nCDase) in S1P generation and inflammatory bowel disease. To this end, we established nCDase expression is increased in patients with ulcerative colitis. Using the dextran sulfate sodium (DSS)-induced colitis model, we determined nCDase activity increased in colon epithelium, but not submucosa, in wild-type (WT) mice. Following DSS, ceramide levels were elevated in colon epithelium from WT and nCDase(-/-) mice, while S1P levels were significantly elevated only in the epithelium of nCDase(-/-) mice. Similarly, cyclooxygenase-2 (Cox-2) levels were significantly elevated only in the epithelium of nCDase(-/-) mice. Neutral CDase(-/-) mice also exhibited higher endotoxin levels in circulation, as well as higher circulating levels of S1P. This increase in S1P in nCDase(-/-) mice was accompanied by a marked leukocytosis, most notably circulating neutrophils and lymphocytes. Taken together these data demonstrate that loss of nCDase results in an unexpected increase in S1P generation in inflammation, and suggests that nCDase may actually protect against inflammation.

Translational landscape of glioblastoma immunotherapy for physicians: guiding clinical practice with basic scientific evidence.

Despite recent advances in cancer therapeutics, glioblastoma (GBM) remains one of the most difficult cancers to treat in both the primary and recurrent settings. GBM presents a unique therapeutic challenge given the immune-privileged environment of the brain and the aggressive nature of the disease. Furthermore, it can change phenotypes throughout the course of disease-switching between mesenchymal, neural, and classic gene signatures, each with specific markers and mechanisms of resistance. Recent advancements in the field of immunotherapy-which utilizes strategies to reenergize or alter the immune system to target cancer-have shown striking results in patients with many types of malignancy. Immune checkpoint inhibitors, adoptive cellular therapy, cellular and peptide vaccines, and other technologies provide clinicians with a vast array of tools to design highly individualized treatment and potential for combination strategies. There are currently over 80 active clinical trials evaluating immunotherapies for GBM, often in combination with standard secondary treatment options including re-resection and anti-angiogenic agents, such as bevacizumab. This review will provide a clinically focused overview of the immune environment present in GBM, which is frequently immunosuppressive and characterized by M2 macrophages, T cell exhaustion, enhanced transforming growth factor-ß signaling, and others. We will also outline existing immunotherapeutic strategies, with a special focus on immune checkpoint inhibitors, chimeric antigen receptor therapy, and dendritic cell vaccines. Finally, we will summarize key discoveries in the field and discuss currently active clinical trials, including combination strategies, burgeoning technology like nucleic acid and nanoparticle therapy, and novel anticancer vaccines. This review aims to provide the most updated summary of the field of immunotherapy for GBM and offer both historical perspective and future directions to help inform clinical practice.

Deletion of GRK1 causes retina degeneration through a transducin-independent mechanism.

Rpe65(-/-) mice are unable to produce 11-cis-retinal, the chromophore of visual pigments. Consequently, the pigment is present as the apoprotein opsin with a minute level of pigment containing 9-cis-retinal as chromophore. Notably, a 10-20% fraction of this opsin is mono-phosphorylated independently of light conditions. To determine the role of rhodopsin kinase (GRK1) in phosphorylating this opsin and to test whether eliminating this phosphorylation would accelerate photoreceptor degeneration, we generated the Rpe65(-/-)Grk1(-/-) mouse. The retinae of Rpe65(-/-)Grk1(-/-) mice had negligible opsin phosphorylation, extensive degeneration with decreased opsin levels, and diminished light-evoked rod responses relative to Rpe65(-/-) mice. These data show that opsin phosphorylation in the Rpe65(-/-) mouse is due to the action of GRK1 and is neuroprotective. However, despite the higher activity of unphosphorylated opsin, the severe loss of opsin in the rapidly degenerating Rpe65(-/-)Grk1(-/-) mice resulted in lower overall opsin activity and in higher rod sensitivity compared with Rpe65(-/-) mice. In Rpe65(-/-)Grk1(-/-)Gnat1(-/-) mice where transduction activation was blocked, degeneration was only partially prevented. Therefore, increased opsin activity in the absence of phosphorylation was not the only mechanism for the accelerated retinal degeneration. Finally, the deletion of GRK1 triggered retinal degeneration in Grk1(-/-) mice after 1 month, even in the absence of apo-opsin. This degeneration was independent of light conditions and occurred even in the absence of transducin in Grk1(-/-)Gnat1(-/-) mice. Taken together, our results demonstrate a light-independent mechanism for retinal degeneration in the absence of GRK1, suggesting a second, not previously recognized role for that kinase.