Nucleocytoplasmic trafficking is essential for BAK1‐ and BKK1‐mediated cell‐death control

BRI1‐ASSOCIATED KINASE 1 (BAK1) was initially identified as a co‐receptor of the brassinosteroid (BR) receptor BRI1. Genetic analyses also revealed that BAK1 and its closest homolog BAK1‐LIKE 1 (BKK1) regulate a BR‐independent cell‐death control pathway. The double null mutant bak1 bkk1 displays a salicylic acid‐ and light‐dependent cell‐death phenotype even without pathogen invasion. Molecular mechanisms of the spontaneous cell death mediated by BAK1 and BKK1 remain unknown. Here we report our identification of a suppressor of bak1 bkk1 (sbb1–1). Genetic analyses indicated that cell‐death symptoms in a weak double mutant, bak1–3 bkk1–1, were completely suppressed by the loss‐of‐function mutation in SBB1, which encodes a nucleoporin (NUP) 85‐like protein. Genetic analyses also demonstrated that individually knocking out three other nucleoporin genes from the SBB1‐located sub‐complex was also able to rescue the cell‐death phenotype of bak1–3 bkk1–1. In addition, a DEAD‐box RNA helicase, DRH1, was identified in the same protein complex as SBB1 via a proteomic approach. The drh1 mutation also rescues the cell‐death symptoms of bak1–3 bkk1–1. Further analyses indicated that export of poly(A)⁺ RNA was greatly blocked in the nup and drh1 mutants, resulting in accumulation of significant levels of mRNAs in the nuclei. Over‐expression of a bacterial NahG gene to inactivate salicylic acid also rescues the cell‐death phenotype of bak1–3 bkk1–1. Mutants suppressing cell‐death symptoms always showed greatly reduced salicylic acid contents. These results suggest that nucleocytoplasmic trafficking, especially of molecules directly or indirectly involved in endogenous salicylic acid accumulation, is critical in BAK1‐ and BKK1‐mediated cell‐death control.     

Specifying the role of BAK1‐interacting receptor‐like kinase 3 in brassinosteroid signaling

Brassinosteroids (BR) are involved in the control of several developmental processes ranging from root elongation to senescence and adaptation to environmental cues. Thus, BR perception and signaling have to be precisely regulated. One regulator is BRI1‐associated kinase 1 (BAK1)‐interacting receptor‐like kinase 3 (BIR3). In the absence of BR, BIR3 forms complexes with BR insensitive 1 (BRI1) and BAK1. However, the biophysical and energetic requirements for complex formation in the absence of the ligand have yet to be determined. Using computational modeling, we simulated the potential complexes between the cytoplasmic domains of BAK1, BRI1 and BIR3. Our calculations and experimental data confirm the interaction of BIR3 with BAK1 and BRI1, with the BAK1 BIR3 interaction clearly favored. Furthermore, we demonstrate that BIR3 and BRI1 share the same interaction site with BAK1. This suggests a competition between BIR3 and BRI1 for binding to BAK1, which results in preferential binding of BIR3 to BAK1 in the absence of the ligand thereby preventing the active participation of BAK1 in BR signaling. Our model also suggests that BAK1 and BRI1 can interact even while BAK1 is in complex with BIR3 at an additional binding site of BAK1 that does not allow active BR signaling.     

Competing G protein‐coupled receptor kinases balance G protein and β‐arrestin signaling

Seven‐transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β‐arrestins, whose recruitment to the activated receptor is regulated by G protein‐coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal‐regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT_1AR) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)‐based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well‐established function in the desensitization of G‐protein activation, GRK2 exerts a strong negative effect on β‐arrestin‐dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2‐dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT_1AR, and HEK293 cells expressing other 7TMRs.     

BAK1, an Arabidopsis LRR receptor-like protein kinase,interacts with BRI1 and modulates brassinosteroid signaling

Brassinosteroids regulate plant growth and development through a protein complex that includes the leucine-rich repeat receptor-like protein kinase (LRR-RLK) brassinosteroid-insensitive 1 (BRI1). Activation tagging was used to identify a dominant genetic suppressor of bri1, bak1-1D (bri1-associated receptor kinase 1-1Dominant), which encodes an LRR-RLK, distinct from BRI1. Overexpression of BAK1 results in elongated organ phenotypes, while a null allele of BAK1 displays a semidwarfed phenotype and has reduced sensitivity to brassinosteroids (BRs). BAK1 is a serine/threonine protein kinase, and BRI1 and BAK1 interact in vitro and in vivo. Expression of a dominant-negative mutant allele of BAK1 causes a severe dwarf phenotype, resembling the phenotype of null bri1 alleles. These results indicate BAK1 is a component of BR signaling.     

The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein alpha subunit GPA1 and regulates abscisic acid signaling

Heterotrimeric G proteins composed of alpha, beta, and gamma subunits link ligand perception by G protein-coupled receptors (GPCRs) with downstream effectors, providing a ubiquitous signaling mechanism in eukaryotes. The Arabidopsis thaliana genome encodes single prototypical Galpha (GPA1) and Gbeta (AGB1) subunits, and two probable Ggamma subunits (AGG1 and AGG2). One Arabidopsis gene, GCR1, encodes a protein with significant sequence similarity to nonplant GPCRs and a predicted 7-transmembrane domain structure characteristic of GPCRs. However, whether GCR1 actually interacts with GPA1 was unknown. We demonstrate by in vitro pull-down assays, by yeast split-ubiquitin assays, and by coimmunoprecipitation from plant tissue that GCR1 and GPA1 are indeed physically coupled. GCR1-GPA1 interaction depends on intracellular domains of GCR1. gcr1 T-DNA insertional mutants exhibit hypersensitivity to abscisic acid (ABA) in assays of root growth, gene regulation, and stomatal response. gcr1 guard cells are also hypersensitive to the lipid metabolite, sphingosine-1-phosphate (S1P), which is a transducer of the ABA signal upstream of GPA1. Because gpa1 mutants exhibit insensitivity in aspects of guard cell ABA and S1P responses, whereas gcr1 mutants exhibit hypersensitivity, GCR1 may act as a negative regulator of GPA1-mediated ABA responses in guard cells.     

OST1‐mediated BTF3L phosphorylation positively regulates CBFs during plant cold responses

Cold stress is a major environmental factor that negatively affects plant growth and survival. OST1 has been identified as a key protein kinase in plant response to cold stress; however, little is known about the underlying molecular mechanism. In this study, we identified BTF3 and BTF3L (BTF3‐like), β‐subunits of a nascent polypeptide‐associated complex (NAC), as OST1 substrates that positively regulate freezing tolerance. OST1 phosphorylates BTF3 and BTF3L in vitro and in vivo, and facilitates their interaction with C‐repeat‐binding factors (CBFs) to promote CBF stability under cold stress. The phosphorylation of BTF3L at the Ser50 residue by OST1 is required for its function in regulating freezing tolerance. In addition, BTF3 and BTF3L proteins positively regulate the expression of CBF genes. These findings unravel a molecular mechanism by which OST1‐BTF3‐CBF module regulates plant response to cold stress.     

A non‐canonical rhodopsin‐mediated insulin receptor signaling pathway in retinal photoreceptor neurons

We previously reported a ligand‐independent and rhodopsin‐dependent insulin receptor (IR) neuroprotective signaling pathway in both rod and cone photoreceptor cells, which is activated through protein–protein interaction. Our previous studies were performed with either retina or isolated rod or cone outer segment preparations and the expression of IR signaling proteins were examined. The isolation of outer segments with large portions of the attached inner segments is a technical challenge. Optiprep? density gradient medium has been used to isolate the cells and subcellular organelles, Optiprep? is a non‐ionic iodixanol‐based medium with a density of 1.320 g/mL. We employed this method to examine the expression of IR and its signaling proteins, and activation of one of the downstream effectors of the IR in isolated photoreceptor cells. Identification of the signaling complexes will be helpful for therapeutic targeting in disease conditions.     

The juxtamembrane domains of Arabidopsis CERK1, BAK1, and FLS2 play a conserved role in chitin‐induced signaling

Arabidopsis thaliana CERK1 is an essential receptor‐like kinase in the chitin signal transduction pathway. The juxtamembrane (JM) domain of CERK1 regulates the kinase activity of this receptor. Here we demonstrate that the JM domains of LysM‐RLKs, CERK1, and OsCERK1 play a functionally conserved role in the activation of chitin signaling in Arabidopsis. The C‐termini of the JM domains of both CERK1 and OsCERK1 are indispensable for their function. Moreover, after replacing the JM domain of CERK1 with that of the nonhomologous RLK, BAK1 (CJBa) or FLS2 (CJFl), the chimeric CERK1 receptors maintained their ability to activate chitin signaling in Arabidopsis. Interestingly, the heterologous expression of CJBa and CJFl did not induce cell death in Nicotiana benthamiana leaves. These results suggest that the JM domains of CERK1, BAK1, and FLS2 play a conserved role in chitin signaling via a mechanism not related to sequence homology.     

Polo‐like kinase 1 phosphorylation of G2 and S‐phase‐expressed 1 protein is essential for p53 inactivation during G2 checkpoint recovery

In response to G2 DNA damage, the p53 pathway is activated to lead to cell‐cycle arrest, but how p53 is eliminated during the subsequent recovery process is poorly understood. It has been established that Polo‐like kinase 1 (Plk1) controls G2 DNA‐damage recovery. However, whether Plk1 activity contributes to p53 inactivation during this process is unknown. In this study, we show that G2 and S‐phase‐expressed 1 (GTSE1) protein, a negative regulator of p53, is required for G2 checkpoint recovery and that Plk1 phosphorylation of GTSE1 at Ser 435 promotes its nuclear localization, and thus shuttles p53 out of the nucleus to lead to its degradation during the recovery.     

Regulation of actin function by protein kinase A‐mediated phosphorylation of Limk1

Proper regulation of the cAMP-dependent protein kinase (protein kinase A, PKA) is necessary for cellular homeostasis, and dysregulation of this kinase is crucial in human disease. Mouse embryonic fibroblasts (MEFs) lacking the PKA regulatory subunit Prkar1a show altered cell morphology and enhanced migration. At the molecular level, these cells showed increased phosphorylation of cofilin, a crucial modulator of actin dynamics, and these changes could be mimicked by stimulating the activity of PKA. Previous studies of cofilin have shown that it is phosphorylated primarily by the LIM domain kinases Limk1 and Limk2, which are under the control of the Rho GTPases and their downstream effectors. In Prkar1a^−/− MEFs, neither Rho nor Rac was activated; rather, we showed that PKA could directly phosphorylate Limk1 and thus enhance the phosphorylation of cofilin. These data indicate that PKA is crucial in cell morphology and migration through its ability to modulate directly the activity of LIM kinase.     

Wnt3‐frizzled 1 chimera as a model to study canonical Wnt signaling

Wnt proteins initiate signaling by binding to seven transmembrane spanning receptors of the frizzled (Fz) family together with the members of the low‐density lipoprotein receptor‐related protein (LRP) 5 and 6. A chimera of human Wnt3 and Fz1 receptor was developed that efficiently activated the TCF‐luciferase reporter. Deletion of the cytoplasmic tail and point mutations in the PDZ binding region in the chimera resulted in the loss of Wnt signaling, suggesting a critical role for the Fz cytoplasmic region in Wnt signaling. The Fz CRD is also critical for Wnt signaling, as a deletion of 29 amino acids in the 2nd cysteine loop resulted in the total loss of TCF‐luciferase activation. DKK‐1 protein blocks upregulation of the TCF‐luciferase reporter by the Wnt3–Fz1 chimera, suggesting involvement of LRP in Wnt3–Fz1 signaling. Expression of a Wnt3–Fz1 chimera in C3H10T1/2 cells resulted in the upregulation of alkaline phosphatase activity and inhibition of adipocyte formation, demonstrating that the Wnt3–Fz1 chimera is a potent activator of differentiation of C3H10T1/2 cells into osteoblasts and an inhibitor of their differentiation into the adipocyte lineage. In summary, the Wnt–Fz chimera approach has the potential to better our understanding of the mechanism of Wnt action and its role, particularly in stem cell differentiation. In addition, this methodology can be utilized to identify inhibitors of either Wnt, Fz or interactors of the canonical pathway, which may have potential therapeutic value in the treatment of cancers and other diseases. J. Cell. Biochem. 109: 876–884, 2010. © 2009 Wiley‐Liss, Inc.     

Lens morphogenesis is dependent on Pax6‐mediated inhibition of the canonical Wnt/beta‐catenin signaling in the lens surface ectoderm

Lens formation in mouse is critically dependent on proper development of the retinal neuroectoderm that is located close beneath the head surface ectoderm. Signaling from the prospective retina triggers lens‐specific gene expression in the surface‐ectoderm. Supression of canonical Wnt/β‐catenin signaling in the surface ectoderm is one of the prerequisites for lens development because, as we show here, ectopic Wnt activation in the retina and lens abrogates lens formation. Wnt inhibiton is mediated by signals coming from the retina but its exact mechanism is unknown. We show that Pax6 directly controls expression of several Wnt inhibitors such as Sfrp1, Sfrp2, and Dkk1 in the presumptive lens. In accordance, absence of Pax6 function leads to aberrant canonical Wnt activity in the presumptive lens that subsequently impairs lens development. Thus Pax6 is required for down‐regulation of canonical Wnt signaling in the presumptive lens ectoderm. genesis 48:86–95, 2010. © 2009 Wiley‐Liss, Inc.