Nucleocytoplasmic shuttling of human inositol phosphate multikinase is influenced by CK2 phosphorylation

Human inositol phosphate multikinase (IPMK) is a multifunctional protein in cellular signal transduction, namely, a multispecific inositol phosphate kinase, phosphatidylinositol 3-kinase, and a scaffold within the mTOR-raptor complex. To fulfill these nuclear and cytoplasmic functions, intracellular targeting of IPMK needs to be regulated. We show here that IPMK, which has been considered to be a preferentially nuclear protein, is a nucleocytoplasmic shuttling protein, whose nuclear export is mediated by classical nuclear export receptor CRM1. We identified a functional nuclear export signal (NES) additionally to its previously described nuclear import signal (NLS). Furthermore, we describe a mechanism by which the activity of the IPMK-NLS is controlled. Protein kinase CK2 binds endogenous IPMK and phosphorylates it at serine 284. Interestingly, this phosphorylation can decrease nuclear localization of IPMK cell type specifically. A controlled nuclear import of IPMK may direct its actions either toward nuclear inositol phosphate (InsPx) metabolism or cytoplasmic actions on InsPx, phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P?], as well as mTOR-raptor.     

Nucleocytoplasmic shuttling by human immunodeficiency virus type 1 Vpr

Human immunodeficiency virus type 1 (HIV-1) is capable of infecting nondividing cells such as macrophages because the viral preintegration complex is able to actively traverse the limiting nuclear pore due to the redundant and possibly overlapping nuclear import signals present in Vpr, matrix, and integrase. We have previously recognized the presence of at least two distinct and novel nuclear import signals residing within Vpr that, unlike matrix and integrase, bypass the classical importin alpha/beta-dependent signals and do not require energy or a RanGTP gradient. We now report that the carboxy-terminal region of Vpr (amino acids 73 to 96) contains a bipartite nuclear localization signal (NLS) composed of multiple arginine residues. Surprisingly, when the leucine-rich Vpr(1-71) fragment, previously shown to harbor an NLS, or full-length Vpr is fused to the C terminus of a green fluorescent protein-pyruvate kinase (GFP-PK) chimera, the resultant protein is almost exclusively detected in the cytoplasm. However, the addition of leptomycin B (LMB), a potent inhibitor of CRM1-dependent nuclear export, produces a shift from a cytoplasmic localization to a nuclear pattern, suggesting that these Vpr fusion proteins shuttle into and out of the nucleus. Studies of nuclear import with GFP-PK-Vpr fusion proteins in the presence of LMB reveals that both of the leucine-rich alpha-helices are required for effective nuclear uptake and thus define a unique NLS. Using a modified heterokaryon analysis, we have localized the Vpr nuclear export signal to the second leucine-rich helix, overlapping a portion of the amino-terminal nuclear import signal. These studies thus define HIV-1 Vpr as a nucleocytoplasmic shuttling protein.     

The activation of the Arabidopsis P-ATPase 1 by the brassinosteroid receptor BRI1 is independent of threonine 948 phosphorylation

The plasma membrane-spanning receptor brassinosteroid insenstive 1 (BRI1) rapidly induces plant cell wall expansion in response to brassinosteroids such as brassinolide (BL). Wall expansion is accompanied by a rapid hyperpolarization of the plasma membrane, which is recordable by measuring the fluorescence lifetime (FLT) of the green fluorescent protein (GFP) fused to BRI1. For the BL induction of hyperpolarization and wall expansion, the activation of the plasma membrane P-type H⁺-ATPase is necessary. Furthermore, the activation of the P-ATPase requires BRI1 kinase activity and appears to be mediated by a BL-modulated association of BRI1 with the proton pump. Here, we show that BRI1 also associates with a mutant version of the Arabidopsis P-ATPase 1 (AHA1) characterized by an exchange of a well-known regulatory threonine for a non-phosphorylatable residue in the auto-inhibitory C-terminal domain. Even more important, BRI1 is still able to activate this AHA1 mutant in response to BL. This suggests a novel mechanism for the enzymatic activation of the P-ATPase by BRI1 in the plasma membrane. Furthermore, we demonstrate that the FLT of BRI1-GFP can be used as a non-invasive probe to analyze long-distance BL signaling in Arabidopsis seedlings.Key words: BRI1, fluorescence lifetime, membrane potential, P-ATPase, cell wall expansionUsing spectro-microscopic technologies, we recently started the quantitative analysis of the properties and subcellular function of GFP fusion of the plasma membrane-localized brassinosteroid (BR) receptor, BRI1, in living plant cells of Arabidopsis thaliana and tobacco (Nicotiana benthamiana) leaf cells.^1,2 Brassinosteroids, such as brassinolide (BL), are involved in responses to biotic and abiotic stresses and developmental processes, including cell elongation.³ The present model of the BR response pathway includes the binding of BRs to BRI1, resulting in the autophosphorylation of the receptor and the subsequent recruitment of the co-receptor BRI1-associated receptor kinase 1 (BAK1). This association is followed by trans-phosphorylation between BRI1 and BAK1 and results in the activation of downstream BR signaling processes leading to differential gene expression and, finally, to the execution of the specific responses.⁴ However, the molecular events that take place in the plasma membrane immediately after the perception of BL and initiate cell elongation still have to be included in this model.⁵ We recently reported a rapid BRI1-GFP-dependent cell wall expansion in Arabidopsis seedlings, which is attributed to wall loosening and water incorporation into the wall, and precedes cell elongation.^1,2 This expansion response was accompanied by a change in the FLT of BRI1-GFP, which reflects an alteration in the plasma membrane potential (E_m).^2,6 For both the FLT change in BRI1-GFP and the wall expansion, the activity of the plasma membrane P-ATPase is crucial. Notably, H⁺-pump activation was shown to depend on the kinase activity of BRI1.² This suggests a fast BRI1-dependent response pathway in the plasma membrane which links BL perception via P-ATPase activation and E_m hyperpolarization to wall expansion. In this report, we demonstrate that the phosphorylation of a conserved threonine in the auto-inhibitory domain of AHA1 is not required for the enzymatic activation by BRI1 suggesting a novel mechanism by which BRI1 may initiate the activation of the P-ATPase. Furthermore, we show that the FLT of BRI1-GFP is a useful and senstitive probe for the non-invasive analysis of systemic signaling processes in living plants.     

Nucleocytoplasmic shuttling of the splicing factor SIPP1

SIPP1 (splicing factor that interacts with PQBP1 and PP1) is a widely expressed protein of 70 kDa that has been implicated in pre-mRNA splicing. It interacts with protein Ser/Thr phosphatase-1 (PP1) and with the polyglutamine-tract-binding protein 1 (PQBP1), which contributes to the pathogenesis of X-linked mental retardation and neurodegenerative diseases caused by polyglutamine tract expansions. We show here that SIPP1 is a nucleocytoplasmic shuttling protein. Under basal circumstances SIPP1 was largely nuclear, but it accumulated in the cytoplasm following UV- or X-radiation. Nuclear import was mediated by two nuclear localization signals. In addition, SIPP1 could be piggy-back transported to the nucleus with its ligand PQBP1. In the nucleus SIPP1 and PQBP1 formed inclusion bodies similar to those detected in polyglutamine diseases. SIPP1 did not function as a nuclear targeting subunit of PP1 but re-localized nuclear PP1 to storage sites for splicing factors. The C-terminal residues of SIPP1, which do not conform to a classic nuclear export signal, were required for its nuclear export via the CMR-1 pathway. Finally, SIPP1 activated pre-mRNA splicing in intact cells, and the extent of splicing activation correlated with the nuclear concentration of SIPP1. We conclude that SIPP1 is a positive regulator of pre-mRNA splicing that is regulated by nucleocytoplasmic shuttling. These findings also have potential implications for a better understanding of the pathogenesis of X-linked mental retardation and polyglutamine-linked neurodegenerative disorders.     

Interdependency of brassinosteroid and auxin signaling in Arabidopsis

How growth regulators provoke context-specific signals is a fundamental question in developmental biology. In plants, both auxin and brassinosteroids (BRs) promote cell expansion, and it was thought that they activated this process through independent mechanisms. In this work, we describe a shared auxin:BR pathway required for seedling growth. Genetic, physiological, and genomic analyses demonstrate that response from one pathway requires the function of the other, and that this interdependence does not act at the level of hormone biosynthetic control. Increased auxin levels saturate the BR-stimulated growth response and greatly reduce BR effects on gene expression. Integration of these two pathways is downstream from BES1 and Aux/IAA proteins, the last known regulatory factors acting downstream of each hormone, and is likely to occur directly on the promoters of auxin:BR target genes. We have developed a new approach to identify potential regulatory elements acting in each hormone pathway, as well as in the shared auxin:BR pathway. We show that one element highly overrepresented in the promoters of auxin- and BR-induced genes is responsive to both hormones and requires BR biosynthesis for normal expression. This work fundamentally alters our view of BR and auxin signaling and describes a powerful new approach to identify regulatory elements required for response to specific stimuli.     

Nucleocytoplasmic shuttling of p53 is essential for MDM2-mediated cytoplasmic degradation but not ubiquitination

As a shuttling protein, p53 is constantly transported through the nuclear pore complex. p53 nucleocytoplasmic transport is carried out by a bipartite nuclear localization signal (NLS) located at its C-terminal domain and two nuclear export signals (NES) located in its N- and C-terminal regions, respectively. The role of nucleocytoplasmic shuttling in p53 ubiquitination and degradation has been a subject of debate. Here we show that the two basic amino acid groups in the p53 bipartite NLS function collaboratively to import p53. Mutations disrupting individual amino acids in the NLS, although causing accumulation of p53 in the cytoplasm to various degrees, reduce but do not eliminate the NLS activity, and these mutants remain sensitive to MDM2 degradation. However, disrupting both parts of the bipartite NLS completely blocks p53 from entering the nucleus and causes p53 to become resistant to MDM2-mediated degradation. Similarly, mutations disrupting four conserved hydrophobic amino acids in the p53 C-terminal NES block p53 export and prohibit it from MDM2 degradation. We also show that colocalization of a nonshuttling p53 with MDM2 either in the nucleus or in the cytoplasm is sufficient for MDM2-induced p53 polyubiquitination but not degradation. Our data provide new insight into the mechanism and regulation of p53 nucleocytoplasmic shuttling and degradation.     

Nuclear-localized BZR1 mediates brassinosteroid-induced growth and feedback suppression of brassinosteroid biosynthesis

Plant steroid hormones, brassinosteroids (BRs), are perceived by a cell surface receptor kinase, BRI1, but how BR binding leads to regulation of gene expression in the nucleus is unknown. Here we describe the identification of BZR1 as a nuclear component of the BR signal transduction pathway. A dominant mutation bzr1-1D suppresses BR-deficient and BR-insensitive (bri1) phenotypes and enhances feedback inhibition of BR biosynthesis. BZR1 protein accumulates in the nucleus of elongating cells of dark-grown hypocotyls and is stabilized by BR signaling and the bzr1-1D mutation. Our results demonstrate that BZR1 is a positive regulator of the BR signaling pathway that mediates both downstream BR responses and feedback regulation of BR biosynthesis.     

Nucleocytoplasmic shuttling of bovine herpesvirus 1 UL47 protein in infected cells

Previous studies with transfected cells have shown that the herpes simplex virus type 1 (HSV-1) and bovine herpesvirus 1 (BHV-1) UL47 proteins shuttle between the nucleus and the cytoplasm. HSV-1 UL47 has also been shown to bind RNA. Here we examine the BHV-1 UL47 protein in infected cells using a green fluorescent protein-UL47-expressing virus. We show that UL47 is detected in the nucleus early in infection. We use fluorescence loss in photobleaching to show that nuclear UL47 undergoes rapid nucleocytoplasmic shuttling. Furthermore, we demonstrate that actinomycin D inhibits the reaccumulation of UL47 in the nuclei of infected cells. These results suggest that UL47 exhibits behavior similar to that of previously characterized RNA-transporting proteins.     

Arabidopsis RTE1 is essential to ethylene receptor ETR1 amino-terminal signaling independent of CTR1

The Arabidopsis (Arabidopsis thaliana) ethylene receptor Ethylene Response1 (ETR1) can mediate the receptor signal output via its carboxyl terminus interacting with the amino (N) terminus of Constitutive Triple Response1 (CTR1) or via its N terminus (etr1^1-349 or the dominant ethylene-insensitive etr1-1^1-349) by an unknown mechanism. Given that CTR1 is essential to ethylene receptor signaling and that overexpression of Reversion To Ethylene Sensitivity1 (RTE1) promotes ETR1 N-terminal signaling, we evaluated the roles of CTR1 and RTE1 in ETR1 N-terminal signaling. The mutant phenotype of ctr1-1 and ctr1-2 was suppressed in part by the transgenes etr1^1-349 and etr1-1^1-349, with etr1-1^1-349 conferring ethylene insensitivity. Coexpression of 35S:RTE1 and etr1^1-349 conferred ethylene insensitivity in ctr1-1, whereas suppression of the ctr1-1 phenotype by etr1^1-349 was prevented by rte1-2. Thus, RTE1 was essential to ETR1 N-terminal signaling independent of the CTR1 pathway. An excess amount of the CTR1 N terminus CTR1^7-560 prevented ethylene receptor signaling, and the CTR1^7-560 overexpressor CTR1-Nox showed a constitutive ethylene response phenotype. Expression of the ETR1 N terminus suppressed the CTR1-Nox phenotype. etr1^1-349 restored the ethylene insensitivity conferred by dominant receptor mutant alleles in the ctr1-1 background. Therefore, ETR1 N-terminal signaling was not mediated by full-length ethylene receptors; rather, full-length ethylene receptors acted cooperatively with the ETR1 N terminus to mediate the receptor signal independent of CTR1. ETR1 N-terminal signaling may involve RTE1, receptor cooperation, and negative regulation by the ETR1 carboxyl terminus.The gaseous plant hormone ethylene is perceived by a small family of ethylene receptors. Arabidopsis (Arabidopsis thaliana) has five ethylene receptors that are structurally similar to prokaryotic two-component histidine kinase (HK) proteins. Mutants defective in multiple ethylene receptor genes show a constitutive ethylene response phenotype, which indicates a negative regulation of ethylene responses by the receptor genes (Hua and Meyerowitz, 1998).The receptor N terminus has three or four transmembrane domains that bind ethylene. The GAF (for cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) domain, which follows the transmembrane helices, mediates noncovalent receptor heterodimerization and may have a role in receptor cooperation (Gamble et al., 2002; O’Malley et al., 2005; Xie et al., 2006; Gao et al., 2008). The subfamily I receptors Ethylene Response1 (ETR1) and Ethylene Response Sensor1 (ERS1) have a conserved HK domain following the GAF domain. For subfamily II members ETR2, Ethylene Insensitive4 (EIN4), and ERS2, the HK domain is less conserved, and they lack most signature motifs essential for HK activity (Chang et al., 1993; Gamble et al., 1998; Hua et al., 1998; Qu and Schaller, 2004; Xie et al., 2006). Among the five receptors, ETR1, ETR2, and EIN4 have a receiver domain following the HK domain. The ETR1 HK domain may have a role in mediating the receptor signal to downstream components, and the HK activity facilitates the ethylene signaling (Clark et al., 1998; Huang et al., 2003; Hall et al., 2012). The receiver domain can dimerize and could involve receptor cooperation (Müller-Dieckmann et al., 1999). However, differential receptor cooperation occurs between the receiver domain-lacking ERS1 and the other ethylene receptors, which does not support the hypothesis that the domains involve receptor cooperation (Liu and Wen, 2012).Acting downstream of the ethylene receptors is Constitutive Triple Response1 (CTR1), a MEK kinase (mitogen-activated protein kinase kinase kinase) with Ser/Thr kinase activity, and the kinase domain locates at the C terminus. The CTR1 N terminus does not share sequence similarity to known domains and can physically interact with the ethylene-receptor HK domain (Clark et al., 1998; Huang et al., 2003). ctr1 mutants showing attenuated CTR1 kinase activity or the ETR1-CTR1 association exhibit various degrees of the constitutive ethylene-response phenotype. For example, the ctr1-1 and ctr1^btk mutations result from the D694E and E626K substitutions, respectively, in the CTR1 kinase domain, and ctr1-1 shows a stronger ethylene-response phenotype than ctr1^btk, with ctr1-1 having much weaker kinase activity than ctr1^btk (Kieber et al., 1993; Huang et al., 2003; Ikeda et al., 2009). The ctr1-8 mutation results in the G354E substitution that prevents the ETR1-CTR1 association, and the mutant exhibits a constitutive ethylene-response phenotype. Overexpression of the CTR1 N terminus CTR1^7-560, which is responsible for interaction with ethylene receptors, leads to constitutive ethylene responses, possibly by titrating out available ethylene receptors (Kieber et al., 1993; Huang et al., 2003). These studies suggest that CTR1 kinase activity and the interaction of CTR1 with the receptor HK domain may be important to the ethylene receptor signal output in suppressing constitutive ethylene responses.Although the ETR1-CTR1 interaction via the HK domain is essential to the ethylene receptor signal output, evidence suggests that the ETR1 receptor signal output can also be independent of the HK activity or domain. The etr1 ers1 loss-of-function mutant displays extreme growth defects. The etr1[HGG] mutation inactivates ETR1 HK activity, and expression of the getr1[HGG] transgene rescues the etr1 ers1 growth defects, which indicates a lack of association of ETR1 receptor signaling and its kinase activity (Wang et al., 2003). The dominant etr1-1 mutation results in the C65Y substitution and confers ethylene insensitivity (Chang et al., 1993), and the expression of the HK domain-lacking etr1^1-349 and ethylene-insensitive etr1-1^1-349 isoforms partially suppresses the growth defects of etr1 ers1-2. Loss-of-function mutations of subfamily II members do not affect etr1-1^1-349 functions. Therefore, etr1-1^1-349 predominantly cooperates with subfamily I receptors to mediate the ethylene receptor signal output (Xie et al., 2006). Biochemical and transformation studies showing that ethylene receptors can form heterodimers and that each receptor is a component of high-molecular-mass complexes explain how ethylene receptors may act cooperatively (Gao et al., 2008; Gao and Schaller, 2009; Chen et al., 2010).Reversion To Ethylene Sensitivity1 (RTE1), a Golgi/endoplasmic reticulum protein, was isolated from a suppressor screen of the dominant ethylene-insensitive etr1-2 mutation. The cross-species complementation of the rte1-2 loss-of-function mutation by the rice (Oryza sativa) RTE Homolog1 (OsRTH1) suggests a conserved mechanism that modulates the ethylene receptor signaling across higher plant species (Zhang et al., 2012). RTE1 and OsRTH1 overexpression led to ethylene insensitivity in wild-type Arabidopsis but not the etr1-7 loss-of-function mutant, and expression of etr1^1-349 restored ethylene insensitivity with RTE1 overexpression in etr1-7 (Resnick et al., 2006; Zhou et al., 2007; Zhang et al., 2010). Coimmunoprecipitation of epitope-tagged ETR1 and RTE1 and Trp fluorescence spectroscopy revealed the physical interaction of RTE1 and ETR1 (Zhou et al., 2007; Dong et al., 2008, 2010). Therefore, RTE1 may directly promote ETR1 receptor signal output through the ETR1 N terminus, but whether RTE1 has an essential role in ETR1 N-terminal signaling remains to be addressed.Currently, the biochemical nature of the ethylene receptor signal is unknown, and the underlying mechanisms of mediation of the ethylene receptor signal output remain uninvestigated. Genetic and biochemical studies suggest that activation of CTR1 by ethylene receptors may suppress constitutive ethylene responses; upon ethylene binding, the receptors are converted to an inactive state and fail to activate CTR1, and the suppression of ethylene responses by CTR1 is alleviated (Hua and Meyerowitz, 1998; Klee, 2004; Wang et al., 2006; Hall et al., 2007). However, this model does not address how the ETR1 N terminus, which does not have the CTR1-interacting site, mediates the receptor signal to suppress constitutive ethylene responses. The receptor signal of the truncated etr1 isoforms may be mediated by other full-length ethylene receptors and then activate CTR1; alternatively, the ETR1 N-terminal signal may be mediated by a pathway independent of CTR1 (Gamble et al., 2002; Qu and Schaller, 2004; Xie et al., 2006). Results showing that mutants defective in multiple ethylene receptor genes exhibit a more severe ethylene-response phenotype than ctr1 and that ctr1 mutants are responsive to ethylene support the presence of a CTR1-independent pathway (Hua and Meyerowitz, 1998; Cancel and Larsen, 2002; Huang et al., 2003; Liu et al., 2010).In this study, we investigated whether mediation of ETR1 N-terminal signaling is independent of CTR1 and whether RTE1 is essential to the CTR1-independent ETR1 N-terminal signaling. The ETR1 N-terminal signaling was not mediated via other full-length ethylene receptors, but the signal of full-length ethylene receptors could be mediated by the ETR1 N terminus independent of CTR1. The ETR1 C terminus may inhibit ETR1 N-terminal signaling, whereby deletion of the C terminus facilitates N-terminal signaling. We propose a model for the possible modulation of ETR1 receptor signaling.     

Nucleocytoplasmic shuttling of the zinc finger protein EZI Is mediated by importin-7-dependent nuclear import and CRM1-independent export mechanisms

Nucleocytoplasmic translocation constitutes a foundation for nuclear proteins to exert their proper functions and hence for various biological reactions to occur normally in eukaryotic cells. We reported previously that EZI/Zfp467, a 12 zinc finger motif-containing protein, localizes predominantly in the nucleus, yet the underlying mechanism still remains elusive. Here we constructed a series of mutant forms of EZI and examined their subcellular localization. The results delineated a non-canonical nuclear localization signal in the region covering the 9th to the 12th zinc fingers, which was necessary for nuclear accumulation of EZI as well as sufficient to confer nuclear localizing ability to a heterologous protein. We also found that the N-terminal domain of EZI is necessary for its nuclear export, the process of which was not sensitive to the CRM1 inhibitor leptomycin B. An interaction proteomics approach and the following co-immunoprecipitation experiments identified the nuclear import receptor importin-7 as a molecule that associated with EZI and, importantly, short interfering RNA-mediated knockdown of importin-7 expression completely abrogated nuclear accumulation of EZI. Taken together, these results identify EZI as a novel cargo protein for importin-7 and demonstrate a nucleocytoplasmic shuttling mechanism that is mediated by importin-7-dependent nuclear localization and CRM1-independent nuclear export.     

Nucleocytoplasmic shuttling of BID is involved in regulating its activities in the DNA-damage response

The BH3-only BID protein acts as a sentinel to interconnect specific death signals to the core apoptotic pathway. Our previous data demonstrated that BID is important for both S-phase arrest and cell death following DNA damage, and that the cell cycle arrest function is regulated by its phosphorylation by the ATM kinase. We also showed that a portion of cellular BID localizes to the nucleus. Here, we demonstrate that etoposide and ionizing radiation induce the exit of BID from the nucleus and that leptomycin B, a specific inhibitor of the nuclear export receptor CRM1, prevents the nuclear exit of BID. BID carries a nuclear export signal (NES) consensus motif; however, it does not seem to be functional. To examine the importance of BID nuclear export, we targeted BID to the nucleus by fusing it to a strong nuclear localization signal (NLS). NLS-BID is phosphorylated in a similar time course as wild-type BID, but does not exit the nucleus following etoposide treatment. Importantly, introducing NLS-BID into BID(-/-) cells failed to restore S-phase arrest and cell death in response to etoposide. These results implicate BID as a nuclear protein and raise the possibility that nucleocytoplasmic shuttling of BID is involved in regulating its activities in the DNA-damage response.     

Nucleocytoplasmic shuttling of hexokinase II in a cancer cell

In yeast, the hexokinase type II enzyme (HXKII) translocates to the nucleus in the presence of excess glucose, and participates in glucose repression. However, no evidence has suggested a nuclear function for HXKII in mammalian cells. Herein, we present data showing nuclear localization of HXKII in HeLa cells, both by immunocytochemistry and subcellular fractionation. HXKII is extruded from the nucleus, at least in part, by the activity of the exportin 1/CrmA system, as demonstrated by increased nuclear expression and decreased cytoplasmic expression after incubation with leptomycin B, a bacterially-derived exportin inhibitor. Furthermore, cytoplasmic localization of HXKII is dependent on its enzymatic activity, as inhibiting HXKII activity using 2-deoxy-d-glucose (2DG) increased nuclear localization. This effect was more significant in cells incubated in the absence of glucose for 24 h prior to addition of 2DG. Regulated translocation of HXKII to the nucleus of mammalian cells could represent a previously unknown glucose-sensing mechanism.     

Characterization of gravitropic inflorescence bending in brassinosteroid biosynthesis and signaling Arabidopsis mutants

The interaction between the plant hormones, brassinosteroids and auxins has been documented in various processes using a variety of plants and plant parts. In this study, detached inflorescences from brassinosteroid biosynthesis and signaling Arabidopsis mutants were evaluated for their gravitropic bending in response to epibrassinolide (EBR) and indole-3-acetic acid (IAA). EBR supplied to the base of detached inflorescences stimulated gravitropic bending in all BR biosynthetic mutants but there was no effect on the BR signaling mutant or wild type plants. When IAA was supplied to the base of BR mutant inflorescences both natural and EBR-induced gravitropic bending was inhibited. Treatment with the auxin inhibitors also decreased both natural and EBR-induced gravitropic bending. No gravitropic bending was observed when the apical tips of BR mutant inflorescences were removed. IAA treatment to the tips of decapitated BR mutant inflorescences restored gravitropic bending to values observed in the inflorescences with an apical tip, however, EBR applied to the tip had no effect. When decapitated inflorescences from BR mutants were treated with IAA to the base and either gel, EBR or IAA was applied to the tip; there was no gravitropic bending. These results show that brassinosteroids have a role in the gravitropic bending response in Arabidopsis and mutants serve to uncover this hidden contributor.