NDPK2 as a signal transducer in the phytochrome-mediated light signaling

Nucleoside-diphosphate kinase (NDPK) 2 in Arabidopsis has been identified as a phytochrome-interacting protein by using the C-terminal domain of phytochrome A (PhyA) as the bait in yeast two-hybrid screening. The far-red light-absorbing form of phytochrome (Pfr) A stimulates NDPK2 gamma-phosphate exchange activity in vitro. To better understand the multiple functions of NDPK and its role in phytochrome-mediated signaling, we characterized the interaction between phytochrome and NDPK2. Domain studies revealed that PER-ARNT-SIM domain A in the C-terminal domain of phytochrome is the binding site for NDPK2. Additionally, phytochrome recognizes both the NDPK2 C-terminal fragment and the NDPK2 hexameric structure to fulfill its binding. To illustrate the mechanism of how the Pfr form of phytochrome stimulates NDPK2, His-197-surrounding residue mutants were made and tested. Results suggested that the H-bonding with His-197 inside the nucleotide-binding pocket is critical for NDPK2 functioning. The pH dependence profiles of NDPK2 indicated that mutants with different activities from the wild type have different pK(a) values of His-197 and that NDPK2 hyperactive mutants possess lower pK(a) values. Because a lower pK(a) value of His-197 accelerates NDPK2 autophosphorylation and the phospho-transfer between the phosphorylated NDPK2 and its kinase substrate, we concluded that the Pfr form of phytochrome stimulates NDPK2 by lowering the pK(a) value of His-197.     

Rice phytochrome A controls apical hook opening after a single light pulse in transgenic tobacco seedlings

Apical hook opening in tobacco seedlings can be induced by a single red light pulse and this induction can be reverted by a subsequent far-red light pulse. The slow hook opening kinetics and the reversibility of an inductive light pulse even after 8 h of darkness indicate the involvement of stable phytochrome. Compared with wild-type, transgenic BN1 seedlings which overexpress rice phytochrome A exhibit a higher sensitivity to low irradiance red light pulses. Moreover, in BN1 seedlings an inductive red light pulse is only partially reversible even after 30 min, whereas wild-type tobacco seedlings show complete reversibility during the entire hook opening process. The data found show that rice phytochrome A is active in transgenic tobacco seedlings in controlling hook opening and that the introduced rice phytochrome A and the endogenous stable phytochrome behave differently in this response.     

Amplification of phytochrome induced morphogenesis in plants by the cryptic red light signal (CRS)

The regulation of endogenous levels of ascorbic acid in soybean by far-red absorbing form of phytochrome (Pfr) and by cryptic red light signal (CRS) was studied. Cryptic red light signal is produced by red light pre-irradiation of a photoreceptor other than far-red absorbing form of phytochrome (Pfr) and CRS amplifies the action of phytochrome. The endogenous level of ascorbic acid levels enhanced by phytochrome was amplified by CRS. The lifetime of CRS was from 0 to 2 h and the peak of enhancement of ascorbic acid due to CRS was between 16 to 24 h of dark incubation after the end of the treatment. CRS was found to be ineffective on UV-B enhanced endogenous levels of ascorbic acid.Key words: ascorbic acid, cryptic red light signal, glycine max, phytochrome, ultraviolet-BThe phytochrome mediated morphogenesis involves the conversion of Pr [red absorbing form] to Pfr [far-red absorbing form] and the magnitude of the response is dependent on Pfr/P tot ratio established at the end of the irradiation.¹ In broom Sorghum anthocyanin synthesis induced by red light [R₁] is reversible with far-red light. But a second red pulse [R₂] given after the reversal resulted in increased anthocyanin production compared to the first pulse [R₁]. When the red pulse was repeatedly given after every reversal with far-red, the anthocyanin production increased proportionately to the number of previously given pulses.² Thus red pre-treatment induced a change in the cellular physiological state or change in content of a relevant substance[s] which is designated as Cryptic Red Light Signal [CRS] associated with red signal transduction.² CRS was first characterized in detail in Broom Sorghum as Pfr amplifying signal produced by red pre-irradiation. CRS is inactive in the absence of Pfr but enhances the action of Pfr. CRS escapes reversal when the plants are exposed to far-red and is probably produced by a different species of phytochrome, distinct from the conventional reversible phytochrome.³We have investigated whether CRS influences other phytochrome regulated processes in plants in addition to anthocyanin synthesis. We chose another process, the synthesis of endogenous ascorbic acid, which is also regulated by conventional phytochrome.⁴ In soybean, the endogenous level of ascorbic acid is enhanced by conventional far-red reversible form of phytochrome. In addition, an independent UV-B photoreceptor [non reversible with far-red light] also enhances the endogenous synthesis of ascorbic acid in soybean. By using repeated pulses of red light, we have demonstrated that the Cryptic Red Signal is operative in soybean also and it amplifies the red light induced enhancement in the level of ascorbic acid. That CRS is active only in the presence of Pfr is demonstrated by the fact that pre-irradiation with red light is ineffective in amplifying UV-B induced enhancement of ascorbic acid levels. A similar observation on UV-B induced anthocyanin synthesis has been made in Broom Sorghum.² A separate UV-B photoreceptor independent of phytochrome operates in the plants.⁵ Although CRS is presumably produced by pre-irradiation with red light, it does not enhance UV-B induced anthocyanin synthesis or ascorbic acid synthesis in the absence of formation of Pfr by the second red pulse.The life-time of CRS was determined as 6 h in 20°C and 3 h in 24°C grown seedlings of Broom Sorghum with reference to anthocyanin synthesis.² The life-time of CRS determined in soybean seedlings grown at 25°C was upto 1 h.⁶ Since growing seedlings at a low temperature enhanced the effectiveness of CRS in Broom Sorghum, it was concluded that low temperature may either extend the lifetime of CRS or generate higher amount of CRS.² Although the exact nature of CRS is yet to be analyzed, work in our laboratory has established the universal nature of this signal and evidences have been obtained for CRS effect in promoting red light induced hypocotyls inhibition in Cucumber seedlings and also red light induced synthesis of betacyanins in Amaranthus seedlings (submitted for publication).     

SPA1, a component of phytochrome A signal transduction,regulates the light signaling current

Mutations in a component of phytochrome A (phyA)-specific light signal transduction, SPA1, result in enhanced responsiveness of Arabidopsis seedlings to red and far-red light. Here, we have examined the effects of spa1 mutations on the two known modes of phyA function, the high-irradiance responses (HIRs) to continuous irradiation with far-red light and the very-low-fluence responses (VLFRs) to inductive pulses of light that establish only a small proportion of active phyA. spa1 mutants exhibited an enhanced VLFR under hourly pulses of far-red light for hypocotyl growth inhibition, cotyledon unfolding, anthocyanin accumulation, block of greening in subsequent white light and negative regulation of phyB signaling. We provide evidence that the phenotype of spa1 mutants in red light is also caused by an increase in the VLFR. Taken together, our results indicate that light-induced hypocotyl growth inhibition in spa1 mutants is primarily due to a VLFR. While wild-type seedlings required hourly pulses of far-red light to induce a VLFR, infrequent irradiation with far-red pulses (every 12 h) was sufficient to induce a strong VLFR of hypocotyl elongation in spa1 mutants. This shows that the effect of the VLFR was more persistent in spa1 mutants than in the wild type. We, therefore, propose that SPA1 has an important function in reducing the persistence of phyA signaling. spa1 mutations also enhanced the HIRs of anthocyanin accumulation and of phyA-mediated responsivity amplification towards phyB. Thus, our results suggest that spa1 mutations amplify both the phyA-mediated VLFR and the HIR.     

Analysis of type I signal peptidase affinity and specificity for preprotein substrates

Type I signal peptidases (SPases) are membrane-bound endopeptidases responsible for the catalytic cleavage of signal peptides from secretory proteins. Here, we analysed the interaction between a bacterial type I SPase and preprotein substrates using surface plasmon resonance. The use of a home-made biosensor surface based on a mixed self-assembled monolayer of thiols on gold allowed qualitative and kinetic analysis. In vitro binding of purified preproteins to a covalently immobilised bacterial SPase was found to be rather efficient (apparent K(D)=10(-7)-10(-8)M). The signal peptide was shown to be a prerequisite for SPase binding and the nature of the mature part of the preprotein significantly affected SPase binding affinity. The developed biosensor containing immobilised SPase is of great importance for analysis of specificity at substrate binding level and for drug screening. In fact, this is the first report of a membrane protein that was covalently attached to a biosensor surface and that retained binding capacity.     

A novel role for aquaporin-5 in enhancing microtubule organization and stability

Aquaporin-5 (AQP5) is a water-specific channel located on the apical surface of airway epithelial cells. In addition to regulating transcellular water permeability, AQP5 can regulate paracellular permeability, though the mechanisms by which this occurs have not been determined. Microtubules also regulate paracellular permeability. Here, we report that AQP5 promotes microtubule assembly and helps maintain the assembled microtubule steady state levels with slower turnover dynamics in cells. Specifically, reduced levels of AQP5 correlated with lower levels of assembled microtubules and decreased paracellular permeability. In contrast, overexpression of AQP5 increased assembly of microtubules, with evidence of increased MT stability, and promoted the formation of long straight microtubules in the apical domain of the epithelial cells. These findings indicate that AQP5-mediated regulation of microtubule dynamics modulates airway epithelial barrier properties and epithelial function.     

Isolation and characterization of a cDNA-clone coding for potato type A phytochrome

We have isolated and sequenced a cDNA clone encoding the apoprotein of a potato phytochrome. Based on the deduced amino acid sequence, which shows 78% amino acid identity to the Arabidopsis phyA and 50% identity to the Arabidopsis phyB open reading frame, we have classified this cDNA clone as potato phyA phytochrome. The amino acid immediately preceding cysteine 323, which is the homologue of oat cystein 321, to which the chromophore has been shown to be attached, is a tyrosine residue. This contrasts with six other type A phytochrome sequences from both monocots and dicots that encode serine in this position. As already observed in three other cDNAs isolated from dicot species, the potato phyA clone encodes a short open reading frame (13 amino acids) preceding the phyA open reading frame (1123 amino acids), supporting the idea that this type of leader sequence might be involved in the regulated expression of the phytochrome apoprotein. Southern blot analysis revealed a single phyA gene as well as other related phytochrome sequences in the potato genome. phyA mRNA levels varied in different organs and were modulated by white light; in seedlings and sprouts, highest levels of mRNA were detected in the etiolated stage. Upon illumination with white light, mRNA levels decreased to the amount found in leaves of re-etiolated plants. Lowest expression was observed in leaves of plants grown in the light, in tubers irrespective of light treatment, and in roots of plants grown in the dark. In roots of plants grown in the light, elevated levels of phyA mRNA were detected. Using a monoclonal antibody generated against pea phytochrome as an immunochemical probe, the protein was only detectable in protein extracts from etiolated seedlings and sprouts.     

High-irradiance responses induced by far-red light in grass seedlings of the wild type or overexpressing phytochrome A

The occurrence of phytochrome-mediated highirradiance responses (HIR), previously characterised largely in dicotyledonous plants, was investigated in Triticum aestivum L., Zea mays L., Lolium multiflorum Lam. and in both wild-type Oryza sativa L. and in transgenic plants overexpressing oat phytochrome A under the control of a 35S promoter. Coleoptile growth was promoted (maize, ryegrass) or inhibited (wild-type rice) by continuous far-red light (FRc). However, at equal fluences, hourly pulses of far-red light (FRp) were equally effective, indicating that the growth responses to FRc were not true HIR. In contrast, in maize and rice, FRc increased anthocyanin content in the coleoptile in a fluence-rate dependent manner. This response was a true HIR as FRp had reduced effects. In maize, anthocyanin levels were significantly higher under FRc than under continuous red light. In rice, overexpression of phytochrome A increased the inhibition of coleoptile growth and the levels of anthocyanin under FRc but not under FRp or under continuous red light. The effect of FRc was fluence-rate dependent. In light-grown rice, overexpression of phytochrome A reduced leaf-sheath length, impaired the response to supplementary far-red light, but did not affect the response to canopy shade-light. In grasses, typical HIR, i.e. fluence-rate dependent responses showing reciprocity failure, can be induced by FRc. Under FRc, overexpressed phytochrome A operates through this action mode in transgenic rice.Abbreviations FR far-red light - FRc continuous far-red light - FRp pulses of far-red light - HIR high-irradiance responses - LFR low-fluence responses - OPHYA transgenic rice overexpressing oat phytochrome A - Pfr far-red light-absorbing form of phytochrome - phyA phytochrome A - R red light - Rc continuous red light - VLFR very low-fluence responses - WT wildtype We thank Marcelo J. Yanovsky for his help with the photographs and Professor Rodolfo A. Sanchez for providing a reprint of the paper by P.J.A.L. de Lint. This work was supported by grants from UBA (AG041) and Fundacion Antorchas (A-13218/1-15) to J.J.C.     

Deoxygenated phosphorothioate inositol phosphate analogs: synthesis, phosphatase stability, and binding affinity

Inositol phosphates, such as 1D-myo-Inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)], are cellular second messengers with potential roles in cancer prevention and therapy. It typically is difficult to attribute specific pharmacological activity to a single inositol phosphate because they are rapidly metabolized by phosphatases and kinases. In this study, we have designed stable analogs of myo-inositol 4,5-bisphosphate [Ins(4,5)P(2)] and Ins(1,4,5)P(3) that retain the cyclohexane scaffold, but lack hydroxyl groups that might be phosphorylated and have phosphate groups replaced with phosphatase-resistant phosphorothioates. An Ins(1,4,5)P(3) analog, 1D-2,3-dideoxy-myo-inositol 1,4,5-trisphosphorothioate, was synthesized from (-)-quebrachitol, and an Ins(4,5)P(2) analog, 1D-1,2,3-trideoxy-myo-inositol 4,5-bisphosphorothioate, was prepared from cyclohexenol. The Ins(1,4,5)P(3) analog was recognized by Ins(1,4,5)P(3) receptor with a binding constant (K(d)) of 810 nM, compared with 54 nM for the native ligand Ins(1,4,5)P(3), and was resistant to dephosphorylation by alkaline phosphatase under conditions in which Ins(1,4,5)P(3) is extensively hydrolyzed. Analogs developed in this study are potential chemical probes for understanding mechanisms of inositol phosphate actions that may be elucidated by eliciting specific and prolonged activation of the Ins(1,4,5)P(3) receptor.     

Novel interactions of Saccharomyces cerevisiae type 1 protein phosphatase identified by single-step affinity purification and mass spectrometry

The catalytic subunit of Saccharomyces cerevisiae type 1 protein phosphatase (PP1(C)) is encoded by the essential gene GLC7 and is involved in regulating diverse cellular processes. To identify potential regulatory or targeting subunits of yeast PP1(C), we tagged Glc7p at its amino terminus with protein A and affinity-purified Glc7p protein complexes from yeast. The purified proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and identified by peptide mass fingerprint analysis using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. To confirm the accuracy of our identifications, peptides from some of the proteins were also sequenced using high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry. Only four of the Glc7p-associated proteins that we identified (Mhp1p, Bni4p, Ref2p, and Sds22p) have previously been shown to interact with Glc7p, and multiple components of the CPF (cleavage and polyadenylation factor) complex involved in messenger RNA 3'-end processing were present as major components in the Glc7p-associated protein fraction. To confirm the interaction of Glc7p with this complex, we used the same approach to purify and characterize the components of the yeast CPF complex using protein A-tagged Pta1p. Six known components of the yeast (CPF) complex, together with Glc7p, were identified among the Pta1p-associated polypeptides using peptide mass fingerprint analysis. Thus Glc7p is a novel component of the CPF complex and may therefore be involved regulating mRNA 3'-end processing.     

Mutant screen distinguishes between residues necessary for light-signal perception and signal transfer by phytochrome B

The phytochromes (phyA to phyE) are a major plant photoreceptor family that regulate a diversity of developmental processes in response to light. The N-terminal 651-amino acid domain of phyB (N651), which binds an open tetrapyrrole chromophore, acts to perceive and transduce regulatory light signals in the cell nucleus. The N651 domain comprises several subdomains: the N-terminal extension, the Per/Arnt/Sim (PAS)-like subdomain (PLD), the cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) subdomain, and the phytochrome (PHY) subdomain. To define functional roles for these subdomains, we mutagenized an Arabidopsis thaliana line expressing N651 fused in tandem to green fluorescent protein, beta-glucuronidase, and a nuclear localization signal. A large-scale screen for long hypocotyl mutants identified 14 novel intragenic missense mutations in the N651 moiety. These new mutations, along with eight previously identified mutations, were distributed throughout N651, indicating that each subdomain has an important function. In vitro analysis of the spectral properties of these mutants enabled them to be classified into two principal classes: light-signal perception mutants (those with defective spectral activity), and signaling mutants (those normal in light perception but defective in intracellular signal transfer). Most spectral mutants were found in the GAF and PHY subdomains. On the other hand, the signaling mutants tend to be located in the N-terminal extension and PLD. These observations indicate that the N-terminal extension and PLD are mainly involved in signal transfer, but that the C-terminal GAF and PHY subdomains are responsible for light perception. Among the signaling mutants, R110Q, G111D, G112D, and R325K were particularly interesting. Alignment with the recently described three-dimensional structure of the PAS-GAF domain of a bacterial phytochrome suggests that these four mutations reside in the vicinity of the phytochrome light-sensing knot.     

ERECTA controls low light intensity-induced differential petiole growth independent of phytochrome B and cryptochrome 2 action in Arabidopsis thaliana

Plants can respond quickly and profoundly to changes in their environment. Several species, including Arabidopsis thaliana, are capable of differential petiole growth driven upward leaf movement (hyponastic growth) to escape from detrimental environmental conditions. Recently, we demonstrated that the leucine-rich repeat receptor-like Ser/Thr kinase gene ERECTA, explains a major effect Quantitative Trait Locus (QTL) for ethylene-induced hyponastic growth in Arabidopsis. Here, we demonstrate that ERECTA controls the hyponastic growth response to low light intensity treatment in a genetic background dependent manner. Moreover, we show that ERECTA affects low light-induced hyponastic growth independent of Phytochrome B and Cryptochrome 2 signaling, despite that these photoreceptors are positive regulators of low light-induced hyponastic growth.Key words: hyponastic growth, petiole, Arabidopsis, low light, ERECTA, differential growth, phytochrome B, cryptochrome 2Plants must adjust growth and reproduction to adverse environmental conditions. Among the strategies that plants employ to escape from unfavorable conditions is differential petiole growth-driven upward leaf movement, called hyponastic growth. Arabidopsis thaliana is able to exhibit a marked hyponastic response upon flooding, which is triggered by endogenous accumulation of the gaseous phytohormone ethylene.¹ Moreover, a similar response is triggered upon low light intensity perception and in response to supra-optimal temperatures.^2–5 By tilting the leaves to a more vertical position during submergence and shading, the plants restore contact with the atmosphere and light, respectively. The kinetics of the hyponastic growth response induced by the various stimuli is remarkably similar. This led to the hypothesis that shared functional genetic components may be employed to control hyponastic growth. Yet, at least part of the signaling cascades is parallel, as the hormonal control of the response differs between the stimuli. Low light-induced hyponastic growth for example does not require ethylene action.² Whereas the response to heat is antagonized by this hormone.⁵ The abiotic stress hormone abscisic acid (ABA) antagonizes ethylene-induced hyponastic growth and stimulates heat-induced hyponastic growth.^5,6 Moreover, ethylene-induced hyponasty does not involve auxin action⁷ whereas both heat- and low light-induced hyponasty require functional auxin signaling and transport components.^2,5In our recent paper, published in The Plant Journal,⁸ we employed Quantitative Trait Locus (QTL) analysis to identify loci involved in the control of ethylene-induced hyponastic petiole growth. By analyzing induced mutants and by complementation analysis of naturally occurring mutant accessions, we found that the leucine-rich repeat receptor-like Ser/Thr kinase gene ERECTA (ER) is a positive regulator of ethylene-induced hyponastic growth and most likely is causal to one of the identified QTLs. In addition, we demonstrated that the ER dependency is not via ER mediated control of ethylene production or sensitivity.Since low light-induced hyponasty does not require ethylene action,² ER may be part of the proposed shared signaling cascade leading to hyponastic growth where ethylene and low light signals meet. Therefore, we studied low light intensity-induced hyponasty in various erecta mutants. Moreover, natural occurring er mutant accessions complemented with a functional, Col-0 derived, ER allele were tested. The response of Lan-0 (Lan-0; with functional ER) to low light was indistinguishable from the response of Landsberg erecta (Ler) (Fig. 1A). However, complemented Ler (ER-Ler) showed an enhanced response compared to Ler (Fig. 1B). The response of mutant er105 was slightly attenuated compared to the wild type Columbia-0 (Fig. 1C). Mutant er104, however, showed an indistinguishable hyponastic growth phenotype to low light compared to the wild type Wassilewskija-2 (Ws-2) (Fig. 1D). Complementation of the natural occurring erecta mutant accession Vancouver-0 (Van-0) resulted in an enhanced hyponastic growth response to low light (Fig. 1E), whereas this was not the case for Hiroshima-1 (Hir-1) (Fig. 1F). Together, these data suggest that ER acts as positive regulator of low light-induced hyponastic growth and therefore may be part of the shared signaling cascade towards differential petiole growth. Yet, the effect is strongly dependent on the genetic background since the effects were not observed in every accession tested.Open in a separate windowFigure 1ERECTA involvement in low light-induced hyponasty. Effect of exposure to low light (spectral neutral reduction in light intensity from 200 to 20 µmol m⁻² s⁻¹) on the kinetics of hyponastic petiole growth in Arabidopsis thaliana. (A) mutant (circles) Ler and wild type (dashed line) Lan-0, (B) Ler and Ler complemented (ER-; squares) with the Col-0 ERECTA allele (ER-Ler), (C) er105 and Col-0 wild type, (D) er104 and Ws-2 wild type, (E) natural mutant Van-0 and Van-0 complemented with the Col-0 ER allele (ER-Van-0), (F) natural mutant Hir-1 and Hir-1 complemented with the Col-0 ER allele (ER-Hir-1). Petiole angles were measured using time-lapse photography and subsequent image analysis. Data is pairwise subtracted, which corrects for diurnal petiole movement in control conditions. For details on this procedure, growth conditions and materials, transformation protocol, treatments, data acquirement and all analyses see.^1,8 Error bars represent standard errors; n ≥ 12.Phytochrome B (PhyB) and Cryptochrome 2 (Cry2) photoreceptor proteins are required for a full induction of low light-induced hyponastic growth.² We transformed the phyb5 cry2 mutant9 (Ler genetic background) with Col-0 derived ER. This complementation did not restore the ability of phyb5 cry2 to induce hyponastic growth to neither ethylene (data not shown) nor low light conditions (Fig. 2A). Mutant phyb5 cry2 plants have a typical constitutive shade avoidance phenotype, reflected by severely elongated organs. This includes enhanced inflorescence and silique length and thin inflorescences (Fig. 2B-D). Complementation with ER resulted in a significant additional effect on these parameters (Fig. 2B-D). Together, this suggests that ER is not an integral part of PhyB nor Cry2 signaling with respect to (hyponastic) growth. Moreover, PhyB and Cry2 control of plant architecture does not require ER action. Rather, ER seems to mediate growth via genetic interaction with light-reliant growth mechanisms, instead of being downstream of photoreceptor action. Studies on the effects of ER on shade avoidance responses and various hormone responses, including cytokinin and auxin, led to the similar conclusion, suggesting a possible role for ER as a molecular hub coordinating light- and hormone-mediated plant growth.^10,11 One could speculate that ER fine-tunes other (than light) environmental clues with light signaling components. A comparable conclusion was drawn previously for gibberellin (GA) reliant growth mechanisms, as er enhanced the negative effect on plant size of the short internode (shi) mutation12 and er represses the positive effect of the spindly mutation in a GA independent manner.¹³Open in a separate windowFigure 2Effects of ERECTA on light signaling. (A) Effect of exposure to low light (spectral neutral reduction in light intensity from 200 to 20 µmol m⁻² s⁻¹) on the kinetics of hyponastic petiole growth of Ler (dashed lines), the photoreceptor double mutant phyb5 cry2 (circles) and this mutant complemented with the Col-0 ERECTA (ER-phyb cry2; squares). For details see legend Figure 1. (B) Plant height, (C) silique length and (D) inflorescence stem thickness of the above mentioned lines. These parameters were measured when the last flower on the plant developed a silique. Plant height was measured from root/shoot junction to inflorescence top. Stem thickness was measured ∼1 cm above the root/shoot junction with a caliper and silique lengths were measured from representative pedicels in the top ∼10 cm of the main inflorescence stem. Error bars represent standard errors; n ≥ 12. Significance levels; *p < 0.05; **p < 0.01; ***p < 0.001; ns = non significant, by Students t-test.     

Dephosphorylation of Tctex2-related dynein light chain by type 2A protein phosphatase

Maspin is a 42kDa tumor suppressor protein that belongs to the serine protease inhibitor (serpin) family. It inhibits cell motility and invasion in vitro, and tumor growth and metastasis in nude mice; however, maspin's molecular mechanism of action has remained elusive. Maspin contains several tyrosine residues and we hypothesized that phosphorylation of maspin could play a role in its biological function. Our study reveals that maspin is phosphorylated on tyrosine moiety(ies) in normal mammary epithelial cells endogenously expressing maspin. In addition, transfection of the maspin gene, using either a stable or inducible system into maspin-deficient breast cancer cell lines, yields a protein product that is phosphorylated on tyrosine residue(s). Furthermore, recombinant maspin protein can be tyrosine-phosphorylated by the kinase domain from the epidermal growth factor receptor in vitro. These novel observations suggest that maspin, which deviates from the classical serpin, may be an important signal transduction molecule in its phosphorylated form.