Dror Shalitin

Regulation of Arabidopsis cryptochrome 2 by blue-light-dependent phosphorylation.

Cryptochromes are blue/ultraviolet-A light receptors that mediate various light responses in plants and animals. But the initial photochemical reaction of cryptochrome is still unclear. For example, although most photoreceptors are known to undergo light-dependent protein modification such as phosphorylation, no blue-light dependent phosphorylation has been reported for a cryptochrome. Arabidopsis cryptochrome 2 (cry2) mediates light regulation of seedling development and photoperiodic flowering. The physiological activity and cellular level of cry2 protein are light-dependent, and protein–protein interactions are important for cry2 function. Here we report that cry2 undergoes a blue-light-dependent phosphorylation, and that cry2 phosphorylation is associated with its function and regulation. Our results suggest that, in the absence of light, cry2 remains unphosphorylated, inactive and stable; absorption of blue light induces the phosphorylation of cry2, triggering photomorphogenic responses and eventually degradation of the photoreceptor.

Blue Light–Dependent in Vivo and in Vitro Phosphorylation of Arabidopsis Cryptochrome 1

Cryptochromes are photolyase-like blue/UV-A light receptors that regulate various light responses in animals and plants. Arabidopsis cryptochrome 1 (cry1) is the major photoreceptor mediating blue light inhibition of hypocotyl elongation. The initial photochemistry underlying cryptochrome function and regulation remain poorly understood. We report here a study of the blue light–dependent phosphorylation of Arabidopsis cry1. Cry1 is detected primarily as unphosphorylated protein in etiolated seedlings, but it is phosphorylated in plants exposed to blue light. Cry1 phosphorylation increases in response to increased fluence of blue light, whereas the phosphorylated cry1 disappears rapidly when plants are transferred from light to dark. Light-dependent cry1 phosphorylation appears specific to blue light, because little cry1 phosphorylation is detected in seedlings treated with red light or far-red light, and it is largely independent from phytochrome actions, because no phytochrome mutants tested significantly affect cry1 phosphorylation. The Arabidopsis cry1 protein expressed and purified from insect cells is phosphorylated in vitro in a blue light–dependent manner, consistent with cry1 undergoing autophosphorylation. To determine whether cry1 phosphorylation is associated with its function or regulation, we isolated and characterized missense cry1 mutants that express full-length CRY1 apoprotein. Mutant residues are found throughout the CRY1 coding sequence, but none of these inactive cry1 mutant proteins shows blue light–induced phosphorylation. These results demonstrate that blue light–dependent cry1 phosphorylation is closely associated with the function or regulation of the photoreceptor and that the overall structure of cry1 is critical to its phosphorylation.

Arabidopsis Cryptochrome 2 Completes Its Posttranslational Life Cycle in the Nucleus

CRY2 is a blue light receptor regulating light inhibition of hypocotyl elongation and photoperiodic flowering in Arabidopsis thaliana. The CRY2 protein is found primarily in the nucleus, and it is known to undergo blue light–dependent phosphorylation and degradation. However, the subcellular location where CRY2 exerts its function or undergoes blue light–dependent phosphorylation and degradation remains unclear. In this study, we analyzed the function and regulation of conditionally nuclear-localized CRY2. Our results show that CRY2 mediates blue light inhibition of hypocotyl elongation and photoperiodic promotion of floral initiation in the nucleus. Consistent with this result and a hypothesis that blue light–dependent phosphorylation is associated with CRY2 function, we demonstrate that CRY2 undergoes blue light–dependent phosphorylation in the nucleus. CRY2 phosphorylation is required for blue light–dependent CRY2 degradation, but only a limited quantity of CRY2 is phosphorylated at any given moment in seedlings exposed to blue light, which explains why continuous blue light illumination is required for CRY2 degradation. Finally, we showed that CRY2 is ubiquitinated in response to blue light and that ubiquitinated CRY2 is degraded by the 26S proteasome in the nucleus. These findings demonstrate that a photoreceptor can complete its posttranslational life cycle (from protein modification, to function, to degradation) inside the nucleus.

Derepression of the NC80 motif is critical for the photoactivation of Arabidopsis CRY2

Cryptochromes are blue light receptors that regulate photomorphogenesis in plants and the circadian clock in animals and plants. Arabidopsis cryptochrome 2 (CRY2) mediates blue light inhibition of hypocotyl elongation and photoperiodic control of floral initiation. CRY2 undergoes blue light-induced phosphorylation, which was hypothesized to be associated with CRY2 photoactivation. To further investigate how light activates CRY2, we analyzed the physiological activities and phosphorylation of various CRY2 fusion proteins in transgenic plants. Our results showed that an 80-residue motif, referred to as NC80, was sufficient to confer the physiological function of CRY2. The GUS-NC80 fusion protein expressed in transgenic plants is constitutively active but unphosphorylated, suggesting that the blue light-induced CRY2 phosphorylation causes a conformational change to derepress the NC80 motif. Consistent with this hypothesis, the CRY2 C-terminal tail was found to be required for the blue light-induced CRY2 phosphorylation but not for the CRY2 activity. We propose that the PHR domain and the C-terminal tail of the unphosphorylated CRY2 form a “closed” conformation to suppress the NC80 motif in the absence of light. In response to blue light, the C-terminal tail of CRY2 is phosphorylated and electrostatically repelled from the surface of the PHR domain to form an “open” conformation, resulting in derepression of the NC80 motif and signal transduction to trigger photomorphogenic responses.

Pleiotrophin produced by multiple myeloma induces transdifferentiation of monocytes into vascular endothelial cells: a novel mechanism of tumor-induced vasculogenesis

Enhanced angiogenesis is a hallmark of cancer. Pleiotrophin (PTN) is an angiogenic factor that is produced by many different human cancers and stimulates tumor blood vessel formation when it is expressed in malignant cancer cells. Recent studies show that monocytes may give rise to vascular endothelium. In these studies, we show that PTN combined with macrophage colony-stimulating factor (M-CSF) induces expression of vascular endothelial cell (VEC) genes and proteins in human monocyte cell lines and monocytes from human peripheral blood (PB). Monocytes induce VEC gene expression and develop tube-like structures when they are exposed to serum or cultured with bone marrow (BM) from patients with multiple myeloma (MM) that express PTN, effects specifically blocked with antiPTN antibodies. When coinjected with human MM cells into severe combined immunodeficient (SCID) mice, green fluorescent protein (GFP)-marked human monocytes were found incorporated into tumor blood vessels and expressed human VEC protein markers and genes that were blocked by anti-PTN antibody. Our results suggest that vasculogenesis in human MM may develop from tumoral production of PTN, which orchestrates the transdifferentiation of monocytes into VECs.

Vorinostat enhances the antimyeloma effects of melphalan and bortezomib

Objectives:  Examine the antitumor activity of the histone deacetylase inhibitor vorinostat’s antitumor activity against multiple myeloma (MM) using cell lines and a murine xenograft model.

Antimyeloma effects of arsenic trioxide are enhanced by melphalan, bortezomib and ascorbic acid

Arsenic trioxide (ATO) induces apoptosis of malignant plasma cells through multiple mechanisms, including inhibition of DNA binding by nuclear factor kappa‐B, a key player in the development of chemoresistance in multiple myeloma (MM). This activity suggests that ATO may be synergistic when combined with other active antimyeloma drugs. To evaluate this, we examined the antimyeloma effects of ATO alone and in combination with bortezomib, melphalan and ascorbic acid (AA) both in vitro and in vivo using a severe combined immunodeficient (SCID)‐hu murine myeloma model. Marked synergistic antimyeloma effects were demonstrated when human MM Los Angeles xenograft IgG lambda light chain (LAGλ‐1) cells were treated in vitro with ATO and any one of these agents. SCID mice bearing human MM LAGλ‐1 tumours were treated with single‐agent ATO, bortezomib, melphalan, or AA, or combinations of ATO with either bortezomib or melphalan and AA. Animals treated with any of these drugs alone showed tumour growth and increases in paraprotein levels similar to control mice, whereas animals treated with ATO‐containing combinations showed markedly suppressed tumour growth and significantly reduced serum paraprotein levels. These in vitro and in vivo results suggest that addition of ATO to other antimyeloma agents may result in improved outcomes for patients with relapsed or refractory MM.