The leucine-rich repeat (LRR) protein, CaLRR1, interacts with the hypersensitive induced reaction (HIR) protein, CaHIR1, and suppresses cell death induced by the CaHIR1 protein

Leucine-rich repeat proteins (LRRs) function in a number of signal transduction pathways via protein–protein interactions. The gene encoding a small protein of pepper, CaLRR1 , is specifically induced upon pathogen challenge and treatment with pathogen-associated molecular patterns (PAMPs). We identified a pepper hypersensitive induced reaction (CaHIR1) protein that interacts with the LRR domain of the CaLRR1 protein using yeast two-hybrid screening. Ectopic expression of the pepper CaHIR1 gene induces cell death in tobacco and Arabidopsis, indicating that the CaHIR1 protein may be a positive regulator of HR-like cell death. Because transformation is very difficult in pepper plants, we over-expressed CaLRR1 and CaHIR1 in Arabidopsis to determine cellular functions of the two genes. The over-expression of the CaHIR1 gene, but not the CaLRR1 gene, in transgenic Arabidopsis confers disease resistance in response to Pseudomonas syringae infection, accompanied by the strong expression of PR genes, the accumulation of both salicylic acid and H₂O₂, and K⁺ efflux in plant cells. In Arabidopsis and tobacco plants over-expressing both CaHIR1 and CaLRR1 , the CaLRR1 protein suppresses not only CaHIR1 -induced cell death, but also PR gene expression elicited by CaHIR1 via its association with HIR protein. We propose that the CaLRR1 protein functions as a novel negative regulator of CaHIR1-mediated cell death responses in plants.     

Phosphoinositide-dependent protein kinase-1 (PDK1)-independent activation of the protein kinase C substrate, protein kinase D

Phosphoinoisitide dependent kinase l (PDK1) is proposed to phosphorylate a key threonine residue within the catalytic domain of the protein kinase C (PKC) superfamily that controls the stability and catalytic competence of these kinases. Hence, in PDK1-null embryonic stem cells intracellular levels of PKCalpha, PKCbeta1, PKCgamma, and PKCepsilon are strikingly reduced. Although PDK1-null cells have reduced endogenous PKC levels they are not completely devoid of PKCs and the integrity of downstream PKC effector pathways in the absence of PDK1 has not been determined. In the present report, the PDK1 requirement for controlling the phosphorylation and activity of a well characterised substrate for PKCs, the serine kinase protein kinase D, has been examined. The data show that in embryonic stem cells and thymocytes loss of PDK1 does not prevent PKC-mediated phosphorylation and activation of protein kinase D. These results reveal that loss of PDK1 does not functionally inactivate all PKC-mediated signal transduction.     

Yotiao protein, a ligand for the NMDA receptor, binds and targets cAMP-dependent protein kinase II(1)

A yeast two-hybrid screen revealed that regulatory subunits (RII) of PKAII bind the Yotiao protein. Yotiao interacts with the NR1 subunit of the NMDA receptor. A purified C-terminal fragment of Yotiao binds PKAII, via an RII binding site constituted by amino acid residues 1452-1469, with a dissociation constant (K(d)) between 50 and 90 nM in vitro. A stable complex composed of Yotiao, RII and NR1 was immunoprecipitated from whole rat brain extracts. Immunostaining analysis disclosed that Yotiao, RIIbeta and NR1 colocalize in striatal and cerebellar neurons. Co-assembly of Yotiao/PKAII complexes with NR1 subunits may promote cAMP-dependent modulation of NMDA receptor activity at synapses, thereby influencing brain development and synaptic plasticity.     

Mutation of proline residues in the NH(2)-terminal region of the multidrug resistance protein,MRP1 (ABCC1): effects on protein expression,membrane localization,and transport function

The Multidrug Resistance Protein, MRP1 (ABCC1) confers drug resistance and transports organic anions such as leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG). Previous studies showed that portions of the first membrane spanning domain (MSD1) and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. We have replaced 12 prolines in MSD1 and CL3 with alanine and determined the effects of these substitutions on MRP1 expression and transport activity. All singly substituted MRP1-Pro mutants could be expressed in HeLa cells, except MRP1-P104A. The expressed mutants also transported LTC(4) and E(2)17betaG, and their K(m) (LTC(4)) values were similar to wild-type MRP1. Expression of the double mutant MRP1-P42/51A was reduced by >80% although it localized to the plasma membrane and transported organic anions. MRP1 expression was also reduced when the first transmembrane helix (amino acids 37-54) was deleted. In contrast, the phenotypes of the multiply substituted CL3 mutants MRP1-P196/205/207/209A and MRP1-P235/255A were comparable to wild-type MRP1. However, Pro(255)-substituted MRP1 mutants showed reduced immunoreactivity with a monoclonal antibody (MAb) whose epitope is located in CL3. We conclude that certain prolines in MSD1 and CL3 play a role in the expression and structure of MRP1.     

A barley (Hordeum vulgare L.) LEA3 protein, HVA1, is abundant in protein storage vacuoles

The HVA1 protein belongs to the LEA3 group, which is expressed during the late stage of seed maturation. It is also induced by exogenous abscisic acid (ABA) and a variety of environmental stresses in germinating barley (Hordeum vulgare L.). In the present work, the potential role of HVA1 was investigated by studying its tissue distribution and subcellular localization in mature and stressed seeds by immuno-microscopic methods. In the mature seed, HVA1 protein was detected in all tissues except the non-living starchy endosperm. During germination the amount of HVA1 protein decreased but did not totally disappear. Incubation with 100 μM ABA, cold treatment or drought stress dramatically increased HVA1 expression in the germinated seed. In this work, the distribution of a LEA3 group protein was studied in a cereal seed for the first time by immuno-electron microscopy. In the scutellum and aleurone layer, HVA1 was localized both in the cytoplasm and protein storage vacuoles (PSVs). HVA1 protein was found to be threefold more abundant in PSVs than in the cytoplasm of an unstressed seed tissue. The ratio increased with ABA or stress treatments to at least ninefold. The role of HVA1 in PSVs remains unclear: a previously suggested possibility is ion sequestration to prevent precipitation during stress. On the other hand, HVA1 protein could also be degraded in PSVs. HVA1 protein does not have the signal peptide typical of proteins which are glycosylated and targeted into the vacuole via the Golgi complex. Because HVA1 is not glycosylated, it may use an alternative, ER-independent vacuolar pathway, also found in yeast cells.     

Conversion of Bfl-1, an anti-apoptotic Bcl-2 family protein,to a potent pro-apoptotic protein by fusion with green fluorescent protein (GFP)

Human Bfl-1 is an anti-apoptotic Bcl-2 family member. Here, we found that Bfl-1 was converted into a potent death-promoting protein by green fluorescent protein (GFP) fusion with its N-terminus. The transient expression of GFP-Bfl-1 induced cytochrome c release and triggered apoptosis in 293T cells, which depended on the mitochondrial localization of GFP-Bfl-1. Apoptosis induced by GFP-Bfl-1 was significantly blocked by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone, but was not blocked by either Bcl-xL or Bfl-1. Our findings provide a useful model for understanding the structural basis of Bcl-2 family proteins that act in an opposite way despite sharing structural similarity between anti-apoptotic and pro-apoptotic proteins.     

Amyloid beta protein precursor (AbetaPP), but not AbetaPP-like protein 2, is bridged to the kinesin light chain by the scaffold protein JNK-interacting protein 1

Proteolytic processing of amyloid beta protein precursor (AbetaPP) generates peptides that regulate normal cell signaling and are implicated in Alzheimer's disease pathogenesis. AbetaPP processing also occurs in nerve processes where AbetaPP is transported from the cell body by kinesin-I, a microtubule motor composed of two kinesin heavy chain and two kinesin light chain (Klc) subunits. AbetaPP transport is supposedly mediated by the direct AbetaPP-Klc1 interaction. Here we demonstrate that the AbetaPP-Klc1 interaction is not direct but is mediated by JNK-interacting protein 1 (JIP1). The phosphotyrosine binding domain of JIP1 binds the cytoplasmic tail of AbetaPP, whereas the JIP1 C-terminal region interacts with the tetratrico-peptide repeats of Klc1. We also show that JIP1 does not bridge the AbetaPP gene family member AbetaPP-like protein 2, APLP2, to Klc1. These results support a model where JIP1 mediates the interaction of AbetaPP to the motor protein kinesin-I and that this JIP1 function is unique for AbetaPP relative to its family member APLP2. Our data suggest that kinesin-I-dependent neuronal AbetaPP transport, which controls AbetaPP processing, may be regulated by JIP1.     

Protection of cardiac myocytes via delta(1)-opioid receptors, protein kinase C, and mitochondrial K(ATP) channels

The objective of the present study was to investigate the role of delta(1)-opioid receptors in mediating cardioprotection in isolated chick cardiac myocytes and to investigate whether protein kinase C and mitochondrial ATP-sensitive K(+) (K(ATP)) channels act downstream of the delta(1)-opioid receptor in mediating this beneficial effect. A 5-min preexposure to the selective delta(1)-opioid receptor agonist (-)-TAN-67 (1 microM) resulted in less myocyte injury during the subsequent prolonged ischemia compared with untreated myocytes. 7-Benzylidenenaltrexone, a selective delta(1)-opioid receptor antagonist, completely blocked the cardioprotective effect of (-)-TAN-67. Naltriben methanesulfonate, a selective delta(2)-opioid receptor antagonist, had only a slight inhibitory effect on (-)-TAN-67-mediated cardioprotection. Nor-binaltorphimine dihydrochloride, a kappa-opioid receptor antagonist, did not affect (-)-TAN-67-mediated cardioprotection. The protein kinase C inhibitor chelerythrine and the K(ATP) channel inhibitors glibenclamide, a nonselective K(ATP) antagonist, and 5-hydroxydecanoic acid, a mitochondrial selective K(ATP) antagonist, reversed the cardioprotective effect of (-)-TAN-67. These results suggest that the delta(1)-opioid receptor is present on cardiac myocytes and mediates a potent cardioprotective effect via protein kinase C and the mitochondrial K(ATP) channel.     

Yeast Sco1, a protein essential for cytochrome c oxidase function is a Cu(I)-binding protein

Sco1 is a conserved essential protein, which has been implicated in the delivery of copper to cytochrome c oxidase, the last enzyme of the electron transport chain. In this study, we show for the first time that the purified C-terminal domain of yeast Sco1 binds one Cu(I)/monomer. X-ray absorption spectroscopy suggests that the Cu(I) is ligated via three ligands, and we show that two cysteines, present in a conserved motif CXXXC, and a conserved histidine are involved in Cu(I) ligation. The mutation of any one of the conserved residues in Sco1 expressed in yeast abrogates the function of Sco1 resulting in a non-functional cytochrome c oxidase complex. Thus, the function of Sco1 correlates with Cu(I) binding. Data obtained from size-exclusion chromatography experiments with mitochondrial lysates suggest that full-length Sco1 may be oligomeric in vivo.     

Effects of C-terminal modifications of GEC1 protein and gamma-aminobutyric acid type A (GABA(A)) receptor-associated protein (GABARAP), two microtubule-associated proteins, on kappa opioid receptor expression

We demonstrated previously that GEC1, a member of the microtubule-associated protein (MAP) family, bound to the human κ opioid receptor (hKOPR) and promoted hKOPR cell surface expression by facilitating its trafficking along the secretory pathway. GABA(A) receptor-associated protein (GABARAP), a GEC1 analog, also enhanced KOPR expression, but to a lesser extent. The MAP family proteins undergo cleavage of their C-terminal residue(s), and the exposed conserved glycine forms conjugates with phosphatidylethanolamine, which associate with membranes. Here, we examined whether such modifications were required for GEC1 and GABARAP to enhance hKOPR expression. When transiently transfected into CHO or Neuro2A cells, GEC1 and GABARAP were cleaved at the C termini. G116A mutation alone or combined with deletion of Lys(117) in GEC1 (GEC1-A) or Leu(117) in GABARAP (GABARAP-A) blocked their C-terminal cleavage, indicating that the conserved Gly(116) is necessary for C-terminal modification. The two GEC1 mutants enhanced hKOPR expression to similar extents as the wild-type GEC1; however, the two GABARAP mutants did not. Immunofluorescence studies showed that HA-GEC1, HA-GEC1-A, and HA-GABARAP were distributed in a punctate manner and co-localized with KOPR-EGFP in the Golgi apparatus, whereas HA-GABARAP-A did not. Pulldown assay of GST-KOPR-C-tail with HA-GEC1 or HA-GABARAP revealed that GEC1 had stronger association with KOPR-C-tail than GABARAP. These results suggest that because of its stronger binding for hKOPR, GEC1 is able to be recruited by hKOPR sufficiently without membrane association via its C-terminal modification; however, due to its weaker affinity for the hKOPR, GABARAP appears to require C-terminal modifications to enhance KOPR expression.     

Amyloid beta protein precursor is phosphorylated by JNK-1 independent of,yet facilitated by,JNK-interacting protein (JIP)-1

Alzheimer's disease (AD) is genetically linked to the processing of amyloid beta protein precursor (AbetaPP). Aside from being the precursor of the amyloid beta (Abeta) found in plaques in the brains of patients with AD, little is known regarding the functional role of AbetaPP. We have recently reported biochemical evidence linking AbetaPP to the JNK signaling cascade by finding that JNK-interacting protein-1 (JIP-1) binds AbetaPP. In order to study the functional implications of this interaction we assayed the carboxyl-terminal of AbetaPP for phosphorylation. We found that the threonine 668 within the AbetaPP intracellular domain (AID or elsewhere AICD) is indeed phosphorylated by JNK1. We surprisingly found that although JIP-1 can facilitate this phosphorylation, it is not required for this process. We also found that JIP-1 only facilitated phosphorylation of AbetaPP but not of the two other family members APLP1 (amyloid precursor-like protein 1) and APLP2. Understanding the connection between AbetaPP phosphorylation and the JNK signaling pathway, which mediates cell response to stress may have important implications in understanding the pathogenesis of Alzheimer's disease.     

Alix, a novel mouse protein undergoing calcium-dependent interaction with the apoptosis-linked-gene 2 (ALG-2) protein

ALG-2 is a EF hand calcium binding protein with sequence homologies to calmodulin. Vito et al have shown that ALG-2 expression is required for apoptosis following a number of death stimuli,1 although nothing is known about the effectors which underlie ALG-2 function. Here we have used ALG-2 as bait in a yeast two hybrid screen of a mouse brain cDNA library. We found that ALG-2 binds to itself and to a novel protein that we call ALG-2 interacting protein X, Alix. Using co-immunoprecipitation experiments, we confirmed ALG-2/ALG-2 binding and demonstrated that this interaction is calcium independent. ALG-2/Alix interaction was also validated by co-immunoprecipitation, but in this case, the binding was found to be strictly calcium dependent. Alix seems highly conserved throughout evolution since it shows significant homologies to a putative C. elegans protein (YNK-1) and to proteins of A. nidulans (PalA) and S. cerevisiae (BRO1). Alix is a potential regulator or downstream effector of ALG-2 action.     

Fyn binding protein, Fyb, interacts with mammalian actin binding protein, mAbp1

The immune cell specific protein Fyn-T binding protein (Fyb) has been identified as a target of the Yersinia antiphagocytic effector Yersinia outer protein H (YopH), but its role in macrophages is unknown. By using Fyb domains as bait to screen a mouse lymphoma cDNA library, we identified a novel interaction partner, mammalian actin binding protein 1 (mAbp1). We show that mAbp1 binds the Fyb N-terminal via its C-terminally located src homology 3 domain. The interaction between Fyb and mAbp1 is detected in macrophage lysates and the proteins co-localize with F-actin in the leading edge. Hence, mAbp1 is likely to constitute a downstream effector of Fyb involved in F-actin dynamics.