Isolation, characterization and biological activity of a CRF-related diuretic peptide from Periplaneta americana L.

A diuretic peptide (Periplaneta-DP) has been isolated from extracts of whole heads of the cockroach, Periplaneta americana. The purified peptide increases cyclic AMP production and the rate of fluid secretion by isolated Malpighian tubules in vitro. In the fluid secretion assay, the response to native Periplaneta-DP is comparable to that obtained with crude extracts of cockroach corpora cardiaca, and the EC50 lies between 10(-8) and 10(-9) M. The primary structure of Periplaneta-DP was established as a 46-residue amidated peptide: T G S G P S L S I V N P L D V L R Q R L L L E I A R R R M R Q S Q D Q I Q A N R E I L Q T I-NH2. Periplaneta-DP is a further member of the recently established family of CRF-related insect diuretic peptides.     

A new species of Saussurea (Asteraceae) from Tibet and its systematic position based on ITS sequence analysis

A new species of Saussurea , S. erecta S. W. Liu, J. T. Pan & J. Q. Liu sp. nov. , is described from Tibet. It resembles S. kingii but may be distinguished by having distinct stems and glabrous achenes. Saussurea kingii was placed in sect. Pseudoeriocoryne of subgen. Eriocoryne ; this section was circumscribed by acaulescence and an inflorescence with congested capitula surrounded by a rosette of leaves. The discovery of S. erecta with distinct stems, cauline leaves and corymbose capitula blurred the delimitation of sect. Pseudoeriocoryne and suggested that the section may be polyphyletic. Both the close relationship and the significant difference between S. erecta and S. kingii were confirmed by analyses of nrDNA ITS sequences. The resulting phylogenies based on ITS data further suggest that Saussurea sect. Pseudoeriocoryne , as traditionally defined, does not constitute a monophyletic group. The rapid radiation and speciation of Saussurea in the Qinghai-Tibetan Plateau, as inferred from ITS phylogeny, are discussed.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 147 , 349–356.     

Isolation and characterization of proSS1-32, a peptide derived from the N-terminal region of porcine preprosomatostatin

A peptide derived from the N-terminal region of porcine prosomatostatin, proSS1-32, has been purified to homogeneity from extracts of porcine upper intestine. Amino acid analysis revealed that the peptide consists of 32 residues. The complete primary structure was determined as: A P S D P R L R Q F L Q K S L A A A A G K Q E L A K Y F L A E L. This sequence obviously comprises residues 1-32 of porcine prosomatostatin since it is identical to the corresponding sequence in human preprosomatostatin. The postulated cleavage site in porcine prosomatostatin is a Leu-Leu bond between residues 32 and 33, thus confirming previous studies of the processing of the somatostatin precursor in the rat and transgenic mouse.     

Structural basis of thermostability. Analysis of stabilizing mutations in subtilisin BPN'

The crystal structures of two thermally stabilized subtilisin BPN' variants, S63 and S88, are reported here at 1.8 and 1.9 A resolution, respectively. The micromolar affinity calcium binding site (site A) has been deleted (Delta75-83) in these variants, enabling the activity and thermostability measurements in chelating conditions. Each of the variants includes mutations known previously to increase the thermostability of calcium-independent subtilisin in addition to new stabilizing mutations. S63 has eight amino acid replacements: D41A, M50F, A73L, Q206W, Y217K, N218S, S221C, and Q271E. S63 has 75-fold greater stability than wild type subtilisin in chelating conditions (10 mm EDTA). The other variant, S88, has ten site-specific changes: Q2K, S3C, P5S, K43N, M50F, A73L, Q206C, Y217K, N218S, and Q271E. The two new cysteines form a disulfide bond, and S88 has 1000 times greater stability than wild type subtilisin in chelating conditions. Comparisons of the two new crystal structures (S63 in space group P2(1) with A cell constants 41.2, 78.1, 36.7, and beta = 114.6 degrees and S88 in space group P2(1)2(1)2(1) with cell constants 54.2, 60.4, and 82.7) with previous structures of subtilisin BPN' reveal that the principal changes are in the N-terminal region. The structural bases of the stabilization effects of the new mutations Q2K, S3C, P5S, D41A, Q206C, and Q206W are generally apparent. The effects are attributed to the new disulfide cross-link and to improved hydrophobic packing, new hydrogen bonds, and other rearrangements in the N-terminal region.     

Cooperative effect of two surface amino acid mutations (Q252L and E170K) in glucose dehydrogenase from Bacillus megaterium IWG3 on stabilization of its oligomeric state

A thermostable glucose dehydrogenase (GlcDH) mutant of Bacillus megaterium IWG3 harboring the Q252L substitution (Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Biol. Chem. 264:6381-6385, 1989) is stable at pH values above 9, but only in the presence of 2 M NaCl. Another GlcDH mutant exhibiting increased stability at an alkaline pH in the absence of NaCl has been isolated previously (S.-H. Baik, T. Ide, H. Yoshida, O. Kagami, and S. Harayama, Appl. Microbiol. Biotechnol. 61:329-335, 2003). This mutant had two amino acid substitutions, Q252L and E170K. In the present study, we characterized three GlcDH mutants harboring the substitutions Q252L, E170K, and Q252L/E170K under low-salt conditions. The GlcDH mutant harboring two substitutions, Q252L/E170K, was stable, but mutants harboring a single substitution, either Q252L or E170K, were unstable at an alkaline pH. Gel filtration chromatography analyses demonstrated that the oligomeric state of the Q252/E170K enzyme was stable, while the tetramers of the enzymes harboring a single substitution (Q252L or E170K) dissociated into dimers at an alkaline pH. These results indicated that the Q252L and E170K substitutions synergistically strengthened the interaction at the dimer-dimer interface. The crystal structure of the E170K/Q252L mutant, determined at 2.0-angstroms resolution, showed that residues 170 and 252 are located in a hydrophobic cavity at the subunit-subunit interface. We concluded that these residues in the wild-type enzyme have thermodynamically unfavorable effects, while the Q252L and E170K substitutions increase the subunit-subunit interactions by stabilizing the hydrophobic cavity.     

青藏高原东缘不同海拔波缘风毛菊资源分配及花部特征对种子数目和质量的影响

通过采样调查法和烘干称量法检测了青藏高原东缘不同海拔种群波缘风毛菊(Saussurea undulata)花期与果期的形态特征, 研究了其花期与果期的资源分配, 并对花期花部特征对果期种子特征的影响作出分析。结果表明: 1)波缘风毛菊在花期将更多的资源投入繁殖器官, 而在果期增加了对种子的资源投资; 2)波缘风毛菊花期部分花部特征的变异导致其果期产生的种子随海拔升高数量减少、百粒重增加, 因为波缘风毛菊需要在高寒草甸的极端环境下保证每粒种子的存活能力, 因此采取了产生更少但更大种子的生存策略。     

Cooperative Effect of Two Surface Amino Acid Mutations (Q252L and E170K) in Glucose Dehydrogenase from Bacillus megaterium IWG3 on Stabilization of Its Oligomeric State

A thermostable glucose dehydrogenase (GlcDH) mutant of Bacillus megaterium IWG3 harboring the Q252L substitution (Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Biol. Chem. 264:6381-6385, 1989) is stable at pH values above 9, but only in the presence of 2 M NaCl. Another GlcDH mutant exhibiting increased stability at an alkaline pH in the absence of NaCl has been isolated previously (S.-H. Baik, T. Ide, H. Yoshida, O. Kagami, and S. Harayama, Appl. Microbiol. Biotechnol. 61:329-335, 2003). This mutant had two amino acid substitutions, Q252L and E170K. In the present study, we characterized three GlcDH mutants harboring the substitutions Q252L, E170K, and Q252L/E170K under low-salt conditions. The GlcDH mutant harboring two substitutions, Q252L/E170K, was stable, but mutants harboring a single substitution, either Q252L or E170K, were unstable at an alkaline pH. Gel filtration chromatography analyses demonstrated that the oligomeric state of the Q252/E170K enzyme was stable, while the tetramers of the enzymes harboring a single substitution (Q252L or E170K) dissociated into dimers at an alkaline pH. These results indicated that the Q252L and E170K substitutions synergistically strengthened the interaction at the dimer-dimer interface. The crystal structure of the E170K/Q252L mutant, determined at 2.0-Å resolution, showed that residues 170 and 252 are located in a hydrophobic cavity at the subunit-subunit interface. We concluded that these residues in the wild-type enzyme have thermodynamically unfavorable effects, while the Q252L and E170K substitutions increase the subunit-subunit interactions by stabilizing the hydrophobic cavity.     

Two functionally distinct manganese clusters formed by introducing a mutation in the carboxyl terminus of a photosystem II reaction center polypeptide, D1, of the green alga Chlamydomonas reinhardtii

To study the function of the carboxyl-terminal domain of a photosystem II (PSII) reaction center polypeptide, D1, chloroplast mutants of the green alga Chlamydomonas reinhardtii have been generated in which Leu-343 and Ala-344 have been simultaneously or individually replaced by Phe and Ser, respectively. The mutants carrying these replacements individually, L343F and A344S, showed a wild-type phenotype. In contrast, the double mutant, L343FA344S, evolved O2 at only 20-30% of the wild-type rate and was unable to grow photosynthetically. In this mutant, PSII accumulated to 60% of the wild-type level, indicating that the O2-evolving activity per PSII was reduced to approximately half that of the wild-type. However, the amount of Mn atom detected in the thylakoids suggested that a normal amount of Mn cluster was assembled. An investigation of the kinetics of flash-induced fluorescence yield decay revealed that the electron transfer from Q(-)(A) to Q(B) was not affected. When a back electron transfer from Q(-)(A) to a donor component was measured in the presence of 3-(3,4-dichlorophenol)-1,1-dimethylurea, a significantly slower component of the Q(-)(A) oxidation was detected in addition to the normal component that corresponds to the back electron transfer from the Q(-)(A) to the S(2)-state of the Mn cluster. Thermoluminescence measurements revealed that L343FA344S cells contained two functionally distinct Mn clusters. One was equivalent to that of the wild-type, while the other was incapable of water oxidation and was able to advance the transition from the S(1)-state to the S(2)-state. These results suggested that a fraction of the Mn cluster had been impaired by the L343FA344S mutation, leading to decreased O2 evolution. We concluded that the structure of the C-terminus of D1 is critical for the formation of the Mn cluster that is capable of water oxidation, in particular, transition to higher S-states.     

Role of Gln1029 in the photoactivation processes of the LOV2 domain in adiantum phytochrome3

Phototropin (phot) is a blue-light receptor in plants. The molecule has two FMN (flavin mononucleotide)-binding domains named the LOV (light-oxygen-voltage) domain, that is a subset of a PAS (per-arnt-sim) superfamily. Illumination of phot-LOV domains produces a covalent C(4a) flavin-cysteinyl adduct, which is called the S390 intermediate state. According to the crystal structures of the LOV2 domain of Adiantum phytochrome3 (phy3), a fusion protein of phot containing the phytochrome chromophoric domain, in the unphotolyzed and S390 states, and the side chain of Gln1029 switches hydrogen bonds with the FMN chromophore. Gln1029 is the hydrogen-bonding donor of the C(4)=O group of FMN in the unphotolyzed state, whereas Gln1029 is the hydrogen-bonding acceptor of the N(5)-H group of FMN in S390. In this paper, we measured the light-induced structural changes in the Q1029L mutant protein of phy3-LOV2 by means of low-temperature FTIR spectroscopy, and the obtained spectra are compared with those of the wild type. Low-temperature UV-visible spectroscopy of Q1029L detected only one intermediate state, S390, at 77-295 K, as well as the wild type. The C(4)=O stretch of FMN at 1710 cm(-1) is shifted to 1723 cm(-1) in Q1029L, presumably because of the lack of hydrogen bonds between Gln1029 and FMN. Upon formation of S390, the C(4)=O group hydrogen bond is weakened in both wild type and Q1029L. These observations are fully consistent with the X-ray crystal structures of the unphotolyzed and S390 states. On the other hand, the C(4)=O stretch of FMN and amide-I vibrations are temperature-independent in Q1029L, in contrast to wild type, in which highly temperature-dependent FTIR spectra are detected. Amide-I vibrations of Q1029L at room temperature are similar to those of the wild type at 77-150 K but not at room temperature. These facts imply that the Q1029L mutant protein lacks progressive protein structural changes following flavin-cysteinyl adduct formation in the wild type, which eventually alter structures of beta sheet and alpha helix in the protein moiety. Hydrogen-bonding interaction of Gln1029 with the FMN chromophore likely plays an important role in the protein structural changes of phy3-LOV2.     

Validation of the reshaped shared epitope HLA-DRB1 classification in rheumatoid arthritis

Recently, we proposed a classification of HLA-DRB1 alleles that reshapes the shared epitope hypothesis in rheumatoid arthritis (RA); according to this model, RA is associated with the RAA shared epitope sequence (72-74 positions) and the association is modulated by the amino acids at positions 70 and 71, resulting in six genotypes with different RA risks. This was the first model to take into account the association between the HLA-DRB1 gene and RA, and linkage data for that gene. In the present study we tested this classification for validity in an independent sample. A new sample of the same size and population (100 RA French Caucasian families) was genotyped for the HLA-DRB1 gene. The alleles were grouped as proposed in the new classification: S1 alleles for the sequences A-RAA or E-RAA; S2 for Q or D-K-RAA; S3D for D-R-RAA; S3P for Q or R-R-RAA; and X alleles for no RAA sequence. Transmission of the alleles was investigated. Genotype odds ratio (OR) calculations were performed through conditional logistic regression, and we tested the homogeneity of these ORs with those of the 100 first trio families (one case and both parents) previously reported. As previously observed, the S2 and S3P alleles were significantly over-transmitted and the S1, S3D and X alleles were under-transmitted. The latter were grouped as L alleles, resulting in the same three-allele classification. The risk hierarchy of the six derived genotypes was the same: (by decreasing OR and with L/L being the reference genotype) S2/S3P, S2/S2, S3P/S3P, S2/L and S3P/L. The homogeneity test between the ORs of the initial and the replication samples revealed no significant differences. The new classification was therefore considered validated, and both samples were pooled to provide improved estimates of RA risk genotypes from the highest (S2/S3P [OR 22.2, 95% confidence interval 9.9-49.7]) to the lowest (S3P/L [OR 4.4, 95% confidence interval 2.3-8.4]).     

四川菊科植物新记录

报道四川菊科植物分布新记录15种,即白山蒿、韦谷草白背变种、百能葳、小一点红、异羽千里光、无毛蟹甲草、黑紫橐吾、蒜叶婆罗门参、华北鸦葱、毛柄蒲儿根、三尖千里光、华麻花头、棉头风毛菊、灰白风毛菊、丽江蓟。列出这些种的标本引证、地理分布和近缘种的区别等。     

中国风毛菊属一新变种

本文发表了辽宁产风毛菊属一新变种, 疏齿苞乌苏里风毛菊 Saussurea ussuriensis Maxim.var.laxiodontolepis Q.Zh.Hanet Sh. Y.Wang.     

Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania.     

Effects of the Enantiomers of BayK 8644 on the Charge Movement of L-type Ca Channels in Guinea-pig Ventricular Myocytes

The effects of the agonist enantiomer S(-)Bay K 8644 on gating charge of L-type Ca channels were studied in single ventricular myocytes. From a holding potential (Vh) of -40 mV, saturating (250 nm) S(-)Bay K shifted the half-distribution voltage of the activation charge (Q1) vs. V curve -7.5 +/- 0.8 mV, almost identical to the shift produced in the Ba conductance vs. V curve (-7.7 +/- 2 mV). The maximum Q1 was reduced by 1.7 +/- 0.2 nC/microF, whereas Q2 (charge moved in inactivated channels) was increased in a similar amount (1.4 +/- 0.4 nC/microF). The steady-state availability curves for Q1, Q2, and Ba current showed almost identical negative shifts of -14.8 +/- 1.7 mV, -18.6 +/- 5.8 mV, and -15.2 +/- 2.7 mV, respectively. The effects of the antagonist enantiomer R(+)BayK 8644 were also studied, the Q1 vs. V curve was not significantly shifted, but Q1max (Vh = -40 mV) was reduced and the Q1 availability curve shifted by -24.6 +/- 1.2 mV. We concluded that: a) the left shift in the Q1 vs. V activation curve produced by S(-)BayK is a purely agonistic effect; b) S(-)BayK induced a significantly larger negative shift in the availability curve than in the Q1 vs. V relation, consistent with a direct promotion of inactivation; c) as expected for a more potent antagonist, R(+)Bay K induced a significantly larger negative shift in the availability curve than did S(-)Bay K.     

A new definition for the consensus sequence of the peroxisome targeting signal type 2

All organisms except the nematode Caenorhabditis elegans have been shown to possess an import system for peroxisomal proteins containing a peroxisome targeting signal type 2 (PTS2). The currently accepted consensus sequence for this amino-terminal nonapeptide is -(R/K)(L/V/I)X(5)(H/Q)(L/A)-. Some C.elegans proteins contain putative PTS2 motifs, including the ortholog (CeMeK) of human mevalonate kinase, an enzyme known to be targeted by PTS2 to mammalian peroxisomes. We cloned the gene for CeMeK (open reading frame Y42G9A.4) and examined the subcellular localization of CeMeK and of two other proteins with putative PTS2s at their amino termini encoded by the open reading frames D1053.2 and W10G11.11. All three proteins localized to the cytosol, confirming and extending the finding that C.elegans lacks PTS2-dependent peroxisomal protein import. The putative PTS2s of the proteins encoded by D1053.2 and W10G11.11 did not function in targeting to peroxisomes in yeast or mammalian cells, suggesting that the current PTS2 consensus sequence is too broad. Analysis of available experimental data on both functional and nonfunctional PTS2s led to two re-evaluated PTS2 consensus sequences: -R(L/V/I/Q)XX(L/V/I/H)(L/S/G/A)X(H/Q)(L/A)-, describes the most common variants of PTS2, while -(R/K)(L/V/I/Q)XX(L/V/I/H/Q)(L/S/G/A/K)X(H/Q)(L/A/F)-, describes essentially all variants of PTS2. These redefined PTS2 consensus sequences will facilitate the identification of proteins of unknown cellular localization as possible peroxisomal proteins.     

贵州植物区系增补Ⅱ

报道了贵州种子植物地理分布新记录属2个,即獐耳细辛属Hepatica Mill.、扭柄花属Streptopus Michx..新记录种14个,即金佛铁线莲Clematis gratopsis W.T.Wang、川鄂獐耳细辛Hepatica henryi (Oliv.) Steward、网脉唐松草Thalictrum reticulatum Franch.、阴山荠Yinshania acutangula (O.E.Schulz) Y.H.Zhang、大花杠柳Periploca tsiangii D.Fang et H.Z.Ling、钻萼唇柱苣苔Chirita subulatisepala W.T.Wang、南川橐吾Ligularia nanchuanica S.W.Liu、鸢尾叶风毛菊Saussurea romuleifolia Franch.、圆叶风毛菊Saussurea rotundifolia Chen、莲座狗舌草Tephroseris changii B.Nord.、少花黄鹌菜Youngia szechuanica(Soderb.) S.Y.Hu、地杨梅Luzula campestris (L.) DC.、疏花粉条儿菜Aletris laxiflora Bur.et Franch.、小花扭柄花Streptopus parviflorus Franch..新记录变种2个,即扇叶虎耳草Saxifraga rufescens Balf.f.var.flabellifolia C.Y.Wu et J.T.Pan、空心柴胡Bupleurum longicaule Wall.ex DC.var.franchetii H.de Boiss.     

营养和光照对不同生态幅风毛菊属植物幼苗形态可塑性的影响

以青藏高原东部4种不同生态幅的风毛菊属植物为试验对象,研究了不同营养和光照条件对风毛菊幼苗相对生长率、根冠比和比叶面积的可塑性影响.结果表明: 营养和光照条件对4个物种幼苗的3个形态特征变化呈现显著影响并存在一定的交互作用.这些形态特征的可塑性及其对营养和光照变化的响应模式存在一定的种间差异.物种与营养、光照的交互作用对风毛菊属幼苗的根冠比和比叶面积(P<0.001)存在一定的显著影响.4种植物幼苗的平均形态表型可塑性指数的大小与其生态分布相关,风毛菊幼苗的平均表型可塑性指数最大(0.71),是分布生境最多的一个物种.风毛菊属植物形态可塑性与其分布生态幅呈一定正相关关系.     

Active sites of diacylglycerol kinase from Escherichia coli are shared between subunits

We show that residues from different subunits participate in forming the active site of the trimeric membrane protein diacylglycerol kinase (DGK) from Escherichia coli. Five likely active-site mutants were identified: A14Q, N72S, E76L, K94L, and D95N. All five of these mutants possessed significantly impaired catalytic function, without evidence of gross structural alterations as judged by their similar near-UV and far-UV circular dichroism spectra. We found that mixtures of either A14Q or E76L with N72S or K94L possessed much greater activity than the mutant proteins by themselves, suggesting that Ala14 and Glu76 may be on one half-site while Asn72 and Lys94 are on another half-site. Consistent with the shared site model, we also found that (1) peak activity of A14Q and N72S subunit mixtures occur at equimolar concentrations; (2) the maximum activity of the A14Q and N72S mixture was 20% of the wild-type enzyme, in good agreement with the theoretical maximum of 25%; (3) the activity of mutant subunits could not be recovered by mixing with the wild-type subunits; (4) a double mutant, A14Q/N72S, bearing mutations in both putative half-sites was found to inactivate wild-type subunits; (5) the concentration dependence of inactivation by the A14Q/N72S mutant could be well described by a shared site model for a trimeric protein. Unexpectedly, we found that the single mutant D95N behaved in a manner similar to the double mutant, A14Q/N72S, inactivating wild-type subunits. We propose that Asp95 plays a role in more than one active site.     

Serine-71 phosphorylation of rac1 modulates downstream signaling

The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways.