Phosphorylation-mimic Mutant Research Articles

Effect of Phosphorylation on Motile Properties of the Mitotic Kinesin-5 Cin8

The mitotic spindle is a microtubule-based bipolar structure which undergoes a well-defined set of morphological changes while mediating the segregation of duplicated chromosomes. It has been established that spindle morphogenesis is governed, in part, by the activity of molecular motors from the kinesin-5 family of bipolar motors with two pairs of catalytic domains located at the opposite sides of the active complex. Kinesin-5 motors are believed to perform their functions by crosslinking and sliding apart antiparallel microtubules originating from opposite spindle poles. Saccharomyces cerevisiaes cells express two kinesin-5 homologues; Cin8 and Kip1 that overlap in essential mitotic functions such as spindle assembly and anaphase spindle elongation.Previous work from our laboratory indicated that Cin8 is differentially phosphorylated during mid-late anaphase at three cyclin-dependent kinase 1 (Cdk1) specific sites located in its motor domain. Phosphorylation of Cin8 at anaphase, causes its detachment from the spindles, reduces spindle elongation rate and aids in maintaining spindle morphology (Avunie-Masala et al., 2011). To understand how phosphorylation regulates the functions of Cin8, we examined its motile properties by a single-molecule fluorescence motility assay, in which movements of single Cin8-3GFP molecules were observing on fixed microtubules. Since Cin8 was shown to be phosphorylated only during anaphase, we first examined its motile properties in crude extracts of yeast cells arrested in the different points of the cells-cycle. We also compared the motile properties of phosphorylation-deficient and phosphorylation-mimic mutants of Cin8 to the motile properties of the wild type Cin8. We found differences in characteristics of Cin8 motility under the different conditions. Results will be presented.

Serine 727 phosphorylation of STAT3: An early change in mouse hepatocarcinogenesis induced by neonatal treatment with diethylnitrosamine

STAT3 activation is involved in development and progression of hepatocellular carcinoma (HCC). We investigated STAT3 activation during hepatocarcinogenesis induced by neonatal diethylnitrosamine (DEN) treatment in mice. Nuclear accumulation and phosphorylation of STAT3 were detected in altered hepatocyte foci in the early stages as well as adenomas and HCCs in the late stages. Although total STAT3 levels were the same between the hepatic lesions and normal livers, S727-phosphorylated STAT3 was enhanced in adenomas and HCCs, whereas Y705-phosphorylated STAT3 was detected mainly in HCCs. In mouse HCC cell lines, although both S727 and Y705 remained un- or hypophosphorylated under serum-free conditions, fetal bovine serum (FBS) induced strong S727/weak Y705 phosphorylation, STAT3 nuclear accumulation and cell proliferation, whereas IL-6 treatment without FBS caused Y705 phosphorylation without S727 phosphorylation, STAT3 nuclear accumulation or cell proliferation. When HCCs were simultaneously treated with FBS/IL-6, selective suppression of S727 phosphorylation by an MEK inhibitor prevented STAT3 nuclear accumulation and cell proliferation. Furthermore, an S727 phosphorylation-deficient STAT3 mutant (S727A) had a diminished capacity to accumulate in the nucleus when compared with wild-type (WT) or the phosphorylation-mimic mutant (S727D) following treatment with FBS/IL-6. After treatment with FBS/IL-6, the cells expressing the S727A mutant proliferated more slowly than those expressing WT or S727D mutant. In contrast, suppression of Y705 phosphorylation by a JAK inhibitor in the FBS/IL-6 treated cells did not affect STAT3 nuclear accumulation or cell proliferation. Taken together, these data demonstrate that STAT3 activation, mainly through S727 phosphorylation, contributes to the DEN-induced hepatocarcinogenesis at the earliest stages.

Dimerization in the Grb7 Protein

In previous studies, we showed that the tyrosine phosphorylation state of growth factor receptor-bound protein 7 (Grb7) affects its ability to bind to the transcription regulator FHL2 and the cortactin-interacting protein, human HS-1-associated protein-1. Here, we present results describing the importance of dimerization in the Grb7-Src homology 2 (SH2) domain in terms of its structural integrity and the ability to bind phosphorylated tyrosine peptide ligands. A tyrosine phosphorylation-mimic mutant (Y80E-Grb7-SH2) is largely dimerization deficient and binds a tyrosine-phosphorylated peptide representative of the receptor tyrosine kinase (RTK) erbB2 with differing thermodynamic characteristics than the wild-type SH2 domain. Another dimerization-deficient mutant (F99R-Grb7-SH2) binds the phosphorylated erbB2 peptide with similarly changed thermodynamic characteristics. Both Y80E-Grb7-SH2 and F99R-Grb7-SH2 are structured by circular dichroism measurements but show reduced thermal stability relative to the wild type-Grb7-SH2 domain as measured by circular dichroism and nuclear magnetic resonance. It is well known that the dimerization state of RTKs (as binding partners to adaptor proteins such as Grb7) plays an important role in their regulation. Here, we propose the phosphorylation state of Grb7-SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs such as erbB2. In this manner, additional dimerization-dependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways.

NNOS modulates NO–ROS signaling in neurons via phosphorylation at Ser847

Background nNOS produces NO and reactive oxygen species (ROS). nNOS activity is regulated thorough phosphorylation at Ser847. However, the physiological relevance of this phosphorylation remains unclear. In the present study, we aimed to determine the implications of this phosphorylation on NO–ROS signaling in neurons, including 8-nitro-cGMP formation, a downstream molecule of NO–ROS signaling. Method We prepared a phosphorylation-mimic mutant of nNOS (Ser847 to Asp; 847D) and examined the enzyme activities in vitro, including NO and ROS production, NADPH oxidation, and uncoupling efficiency. Furthermore, we generated PC12 cells expressing the nNOS mutant and measured the generation of NO, ROS, and 8-nitro-cGMP in these cells. Next, we determined nicotine-induced ROS and 8-nitro-cGMP generation in cerebellar granule neurons (CGNs). Results In vitro analysis showed that compared to the wild-type (WT) enzyme, the 847D mutant had decreased NO production and increased ROS production and NADPH oxidation. The uncoupling efficiency of the WT and 847D mutant was 49% and 62%, respectively. In vivo analysis showed that 847D-expressing PC12 cells exhibited lower NO production and higher ROS production than WT-expressing PC12 cells. We observed strong immunoreactivity only in nNOS-expressing cells on immunostaining with an 8-nitro-cGMP-specific antibody; the fluorescent intensity in 847D-expressing cells was greater than that in WT-expressing cells. We detected nicotine-induced ROS and 8-nitro-cGMP production in CGNs. These production reduced on pretreatment of the CGNs with a calmodulin kinase II inhibitor. These results suggest that nNOS regulates NO-ROS signaling, including 8-nitro-cGMP production, in neurons via phosphorylation at Ser847.

Phosphorylation and activation of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) by Ca2+/calmodulin-dependent protein kinase I (CaMKI)

Nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N/PPM1E) is an enzyme that dephosphorylates and downregulates multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) as well as AMP-dependent protein kinase. In our previous study, we found that zebrafish CaMKP-N (zCaMKP-N) underwent proteolytic processing and translocated to cytosol in a proteasome inhibitor-sensitive manner. In the present study, we found that zCaMKP-N is regulated by phosphorylation at Ser-480. When zCaMKP-N was incubated with the activated CaMKI, time-dependent phosphorylation of the enzyme was observed. This phosphorylation was significantly reduced when Ser-480 was replaced by Ala, suggesting that CaMKI phosphorylates Ser-480 of zCaMKP-N. Phosphorylation-mimic mutants, S480D and S480E, showed higher phosphatase activities than those of wild type and S480A mutant in solution-based phosphatase assay using various substrates. Furthermore, autophosphorylation of CaMKII after ionomycin treatment was more severely attenuated in Neuro2a cells when CaMKII was cotransfected with the phosphorylation-mimic mutant of zCaMKP-N than with the wild-type or non-phosphorylatable zCaMKP-N. These results strongly suggest that phosphorylation of zCaMKP-N at Ser-480 by CaMKI activates CaMKP-N catalytic activity and thereby downregulates multifunctional CaMKs in the cytosol.

Effects of a Phosphomimetic Mutant of RAP80 on Linear Polyubiquitin Binding Probed by Calorimetric Analysis

RAP80 plays a key role in DNA damage responses by recognizing K63-linked polyubiquitin moieties through its two ubiquitin-interacting motif (UIM) domains. The linker between the two UIMs possesses a phosphorylation site, but the relationship between phosphorylation and polyubiquitin recognition remains elusive. We investigated the interaction between a phosphorylation-mimic RAP80 mutant S101E and linear polyubiquitins, structurally equivalent to the K63-linked ones, using isothermal titration calorimetry (ITC). ITC analysis revealed differential binding affinities for linear tetraubiquitin by otherwise equivalent UIMs in S101E. Mutational analysis supported such differential polyubiquitin recognition by S101E. Our results suggest a potential crosstalk between polyubiquitin recognition and phosphorylation in RAP80.

Structural Transition of N-Terminal Extension of Cardiac Troponin-I Complexed with Troponin-C caused by PKC-Mediated Phosphorylation, as Revealed by Spin Labeling EPR

Cardiac troponin (cTn) complex consisting of cTnI, cTnC and cTnT is responsible for cardiac mechanotransduction. Aberrent phosophorylation of cTnI by PKC on S42/44 or its phosphorylation mimic mutant S42/44D decreases Ca2+ sensitivity and tension force, consequently generating heart failure. Until now, consequence of cTnI by PKC phosphorylation on molecular level and associated structural mechanisms involved in it are still elusive. Combined with previously published NMR spectra and our bioinformatics analysis, this distinct N-extension of cTnI is strongly shown to be intrinsically disordered. To simulate the possible conformation of this intrinsically disordered region and potential conformational transition caused by PKC when binding with cTnC, Monte Carlo simulation was implemented to explore potential docking sites on cTnC, of which all the predicted structures were evaluated by total and binding energy with the criteria under RosettaCommons software suite. We screened around 10,000 structures for cTnC-I and cTnC-I (S42/44D) complexes, respectively. For cTnC-I complex, flexible N-extension of cTnI docked on N-domain of cTnC, putatively assisting to stabilize the full-open state of cTnC which binds strongly the regulatory region of cTnI to initiate muscle contraction. In contrast, as for cTnC-I (S42/44D), it localized on C-domain of cTnC, thus deficiently to maintain full-open state of cTnC, causing a weak interaction between the regulatory region of cTnI (S42/44D) and cTnC to generate heart failure. To verify our prediction, we are measuring residual mobility and inter-residual distance from spin labels placed on cTnI or cTnI (S42/44D) and cTnC. Preliminary mobility measurement of MSL-labeled cTnC(C84) indicated loose interaction between the regulatory region of cTnI (S42/44D) and cTnC. Measurements on the other sites are in progress.

AATYK1A phosphorylation by Cdk5 regulates the recycling endosome pathway

Trafficking of recycling endosomes (REs) is regulated by the small GTPase, Rab11A; however, the regulatory mechanism remains elusive. Apoptosis-associated tyrosine kinase 1A (AATYK1A) is a Ser/Thr kinase expressed highly in brain. We have recently shown that AATYK1A localizes to Rab11A-positive RE and is phosphorylated at Ser34 by cyclin-dependent kinase 5 (Cdk5). Here, we have investigated a role of AATYK1A and its phosphorylation in recycling endosomal trafficking using Chinese hamster ovary-K1 (CHO-K1) cells. AATYK1A localizes predominantly to Rab11A-positive pericentrosomal endocytic recycling compartment (ERC). Phosphorylation at Ser34 of AATYK1A disrupts its accumulation in the pericentrosomal ERC. Consistently, phosphorylation-mimic mutant (AATYK1A-S34D) did not accumulate in the ERC and additionally attenuated ERC formation. ERC formation suppression can be reversed by constitutively active Rab11A-Q70L, suggesting a functional link between AATYK1A phosphorylation and Rab11A activity. Although no direct interaction between AATYK1A and Rab11A could be detected, the exchange of guanine nucleotides bound to Rab11A was significantly reduced in the presence of the phosphorylation-mimic AATYK1A-S34D. Together, our results reveal a regulatory role for AATYK1A in the formation of pericentrosomal ERC. They furthermore indicate that Cdk5 can disrupt ERC formation via Ser34 phosphorylation of AATYK1A. Finally, our data suggest a mechanism by which AATYK1A signaling couples Cdk5 to Rab11A activity.

W1704 Polyamines Enhance Intestinal Epithelial Restitution by Increasing Stim1-Mediated CA 2+ Influx Through Store-Operated CA 2+ Channel After Wounding

gastric epithelial cells as susceptible to both alcohol-induced cell damage (P=0.07) and apoptosis (P=0.06) as the gastric endothelial cells. Conversely, forced overexpression of survivin by transient transfection rendered gastric endothelial cells as resistant to both alcohol-induced cell damage (P=0.08) and apoptosis (P=0.06) as mock-transfected gastric epithelial cells. Moreover, overexpression of a threonine-34 to glutamate phosphorylation mimic mutant survivin construct rendered gastric endothelial cells significantly more resistant to alcohol-induced damage (P<0.02) and apoptosis (P<0.02) vs. mock-transfected gastric epithelial cells. CONCLUSIONS: 1) Disparate survivin expression levels can explain, in part, the discrepancy between gastric epithelial and endothelial cell susceptibility to alcoholinduced injury. 2) A negatively charged amino acid substitution at position 34, or the phosphorylation modification that naturally occurs on threonine-34 producing a negative charge at this position, increases the potency of survivin in mediating protection against alcohol-induced gastric mucosal cellular injury. (Supported by NIH R01AA14946)

AATYK1A phosphorylation by Cdk5 regulates the recycling endosome pathway

Trafficking of recycling endosomes (REs) is regulated by the small GTPase, Rab11A; however, the regulatory mechanism remains elusive. Apoptosis-associated tyrosine kinase 1A (AATYK1A) is a Ser/Thr kinase expressed highly in brain. We have recently shown that AATYK1A localizes to Rab11A-positive RE and is phosphorylated at Ser34 by cyclin-dependent kinase 5 (Cdk5). Here, we have investigated a role of AATYK1A and its phosphorylation in recycling endosomal trafficking using Chinese hamster ovary-K1 (CHO-K1) cells. AATYK1A localizes predominantly to Rab11A-positive pericentrosomal endocytic recycling compartment (ERC). Phosphorylation at Ser34 of AATYK1A disrupts its accumulation in the pericentrosomal ERC. Consistently, phosphorylation-mimic mutant (AATYK1A-S34D) did not accumulate in the ERC and additionally attenuated ERC formation. ERC formation suppression can be reversed by constitutively active Rab11A-Q70L, suggesting a functional link between AATYK1A phosphorylation and Rab11A activity. Although no direct interaction between AATYK1A and Rab11A could be detected, the exchange of guanine nucleotides bound to Rab11A was significantly reduced in the presence of the phosphorylation-mimic AATYK1A-S34D. Together, our results reveal a regulatory role for AATYK1A in the formation of pericentrosomal ERC. They furthermore indicate that Cdk5 can disrupt ERC formation via Ser34 phosphorylation of AATYK1A. Finally, our data suggest a mechanism by which AATYK1A signaling couples Cdk5 to Rab11A activity.

Synapse formation regulated by protein tyrosine phosphatase receptor T through interaction with cell adhesion molecules and Fyn

The receptor-type protein tyrosine phosphatases (RPTPs) have been linked to signal transduction, cell adhesion, and neurite extension. PTPRT/RPTPrho is exclusively expressed in the central nervous system and regulates synapse formation by interacting with cell adhesion molecules and Fyn protein tyrosine kinase. Overexpression of PTPRT in cultured neurons increased the number of excitatory and inhibitory synapses by recruiting neuroligins that interact with PTPRT through their ecto-domains. In contrast, knockdown of PTPRT inhibited synapse formation and withered dendrites. Incubation of cultured neurons with recombinant proteins containing the extracellular region of PTPRT reduced the number of synapses by inhibiting the interaction between ecto-domains. Synapse formation by PTPRT was inhibited by phosphorylation of tyrosine 912 within the membrane-proximal catalytic domain of PTPRT by Fyn. This tyrosine phosphorylation reduced phosphatase activity of PTPRT and reinforced homophilic interactions of PTPRT, thereby preventing the heterophilic interaction between PTPRT and neuroligins. These results suggest that brain-specific PTPRT regulates synapse formation through interaction with cell adhesion molecules, and this function and the phosphatase activity are attenuated through tyrosine phosphorylation by the synaptic tyrosine kinase Fyn.

Phosphorylation of ARD1 by IKKβ contributes to its destabilization and degradation

IκB kinase β (IKKβ), a major kinase downstream of various proinflammatory signals, mediates multiple cellular functions through phosphorylation and regulation of its substrates. On the basis of protein sequence analysis, we identified arrest-defective protein 1 (ARD1), a protein involved in apoptosis and cell proliferation processes in many human cancer cells, as a new IKKβ substrate. We provided evidence showing that ARD1 is indeed a bona fide substrate of IKKβ. IKKβ physically associated with ARD1 and phosphorylated it at Ser209. Phosphorylation by IKKβ destabilized ARD1 and induced its proteasome-mediated degradation. Impaired growth suppression was observed in ARD1 phosphorylation-mimic mutant (S209E)-transfected cells as compared with ARD1 non-phosphorylatable mutant (S209A)-transfected cells. Our findings of molecular interactions between ARD1 and IKKβ may enable further understanding of the upstream regulation mechanisms of ARD1 and of the diverse functions of IKKβ.

Constitutive phosphorylation of a Rac GAP MgcRacGAP is implicated in v‐Src‐induced transformation of NIH3T3 cells

MgcRacGAP plays critical roles in cell division through regulating Rho family small GTPases. As we previously reported, phosphorylation of MgcRacGAP on serine 387 (S387) is induced by Aurora B kinase at the midbody during cytokinesis, which is a critical step of cytokinesis. Phosphorylation of S387-MgcRacGAP converts it from RacGAP to RhoGAP, leading to completion of cytokinesis. Here we show that MgcRacGAP is prominently phosphorylated on S387 even in the interphase of v-Src-transformed NIH3T3 cells in the cytoplasm, but not in the interphase of parental NIH3T3 or H-RasV12-transformed NIH3T3 cells. Interestingly, levels of phosphorylation on S387 (pS387) correlated with soft agar colony-forming abilities of v-Src-transformed NIH3T3 cells. Expression of a phosphorylation-mimic mutant MgcRacGAP-S387D enhanced colony formation of v-Src-transformed NIH3T3 cells. Surprisingly, a Rac1 inhibitor but not kinase inhibitors including Aurora B kinase inhibitor specifically inhibited phosphorylation of S387-MgcRacGAP in v-Src-transformed NIH3T3 cells, suggesting the v-Src-induced pathological positive feedback mechanisms towards Rac1 activation using pS387-MgcRacGAP. These results indicated the difference in the mechanisms between v-Src- and H-RasV12-induced transformation, and should shed some light on pathological roles of disordered phosphorylation of MgcRacGAP at S387 in v-Src-induced cell transformation.

Mutational analysis of the cytoplasmic domain of CEACAM1-4L in humanized mammary glands reveals key residues involved in lumen formation: Stimulation by Thr-457 and inhibition by Ser-461

CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a type I transmembrane glycoprotein involved in cell–cell adhesion, undergoes extensive alternative splicing, resulting in isoforms with 1–4 Ig-like extracellular domains (ECDs) with either long or short cytoplasmic tails. We have previously shown that CEACAM1-4L (4 ECDs with a long cytoplasmic domain) formed glands with lumena in humanized mammary mouse fat pads in NOD/SCID mice. In order to identify the key residues of CEACAM1-4L that play essential roles in lumen formation, we introduced phosphorylation mimic (e.g., Thr-457 or Ser-461 to Asp) or null mutations (Thr-457 or Ser-461 to Ala) into the cytoplasmic domain of CEACAM1-4L and tested them in both the in vivo mouse model and in vitro Matrigel model of mammary morphogenesis. MCF7 cells stably expressing CEACAM1-4L with the single mutation T457D or the double mutant T457D+S461D, but not the null mutants induced central lumen formation in 3D Matrigel and in humanized mammary fat pads. However, the single phosphorylation mimic mutation S461D, but not the null mutation blocked lumen formation in both models, suggesting that S461 has inhibitory function in glandular lumen formation. Compared to our results for the -4S isoform (Chen et al., J. Biol. Chem, 282: 5749–5760, 2008), the T457A null mutation blocks lumen formation for the -4L but not for the -4S isoform. This difference is likely due to the fact that phosphorylation of S459 (absent in the -4L isoform) positively compensates for loss of T457 in the -4S isoform, while S461 (absent in the -4S isoform) negatively regulates lumen formation in the -4L isoform. Thus, phosphorylation of these key residues may exert a fine control over the role of the -4L isoform (compared to the -4S isoform) in lumen formation.

Multisite Phosphorylation of Arabidopsis HFR1 by Casein Kinase II and a Plausible Role in Regulating Its Degradation Rate

Arabidopsis Long Hypocotyl in Far-Red Light 1 (HFR1), a bHLH transcription factor, plays a critical role in promoting seedling photomorphogenesis and in balancing the shade-avoidance response under canopy shade conditions. Previous studies have established that HFR1 protein is degraded in darkness and is stabilized under light conditions to promote light signaling. How light regulates HFR1 stability is not well understood. In this study, we show that Arabidopsis HFR1 can be phosphorylated by recombinant casein kinase II (CKII) and plant extract in vitro and that phosphorylation of HFR1 can be effectively reduced by treatments with two CKII-specific inhibitors, 5,6-dichloro-1-beta-d-ribofuranosyl-benzimidazole (DRB) and heparin. We demonstrate that HFR1 physically interacts with the CKB1 and CKB2 regulatory subunits of CKII. Mutagenesis studies indicate that HFR1 is phosphorylated at multiple serine (Ser) residues in the N-terminal regulatory domain of HFR1. We also show that phosphorylation of HFR1 is promoted by light and that a predicted CKII site, Ser(122), represents a major phosphorylation site of HFR1 under both dark and light conditions. Comparison of wild-type, phosphorylation-deficient, and phosphorylation-mimic mutant proteins suggests that phosphorylation acts to reduce the degradation rate of HFR1. Together, our results suggest that CKII-mediated phosphorylation represents an important post-translational modification influencing the stability and signaling activity of Arabidopsis HFR1.

Negative regulation of Jak2 by its auto-phosphorylation at tyrosine 913 via the Epo signaling pathway

Janus kinase 2 (Jak2) has a pivotal role in erythropoietin (Epo) signaling pathway, including erythrocyte differentiation and Stat5 activation. In the course of screening for critical phosphorylation of tyrosine residues in Jak2, we identified tyrosine 913 (Y 913) as a novel and functional phosphorylation site, which negatively regulates Jak2. Phosphorylation at Y 913 rapidly occurred and was sustained for at least 120 min after Epo stimulation, in contrast to the transient phosphorylation of Y 1007/1008 in the activation loop of Jak2. Interestingly, phosphorylation defective mutation of Y 913 (Y 913F) results in a significant enhancement of Epo-induced Jak2 activation, whereas phosphorylation mimic mutation of Y 913 (Y 913E) completely abrogated its activation. Furthermore, Jak2 deficient fetal liver cells expressing Y 913F mutant generated many mature erythroid BFU-E and CFU-E colonies, while Y 913E mutant failed to reconstitute Jak2 deficiency. We also demonstrate, in Jak1, phosphorylation of Y 939, a corresponding tyrosine residue with Y 913, negatively regulated Jak1 signaling pathway. Accordingly, our results suggest that this tyrosine phosphorylation in JH1 domain may be involved in common negative regulation mechanism for Jak family.

Aurora‐A kinase phosphorylation of Aurora‐A kinase interacting protein (AIP) and stabilization of the enzyme‐substrate complex

Aurora-A is an oncogenic kinase that plays essential roles in mitosis as well as cell survival. Aurora-A interacting protein (AIP) was identified as a negative regulator of Aurora-A with its ectopic over expression inducing destabilization of Aurora-A protein. Here we present evidence that in human cells, contrary to the earlier report, AIP functions in stabilizing rather than destabilizing Aurora-A. Furthermore, AIP is phosphorylated on Serine 70 by Aurora-A but not Aurora-B and expression of phosphorylation mimic mutant of AIP results in prolonged protein stability compared to unphosphorylatable mutant. We observed that when co-expressed with AIP, protein levels of both Aurora-A and Aurora-B are markedly elevated regardless of their kinase activities and phosphorylation state of AIP. Interaction of Aurora kinases with AIP is necessary for this elevated stability. This phenomenon is commonly detected in several human cancer cell lines used in this study. Depletion of AIP by RNA interference decreased Aurora-A but not Aurora-B in two of the three cell lines analyzed, indicating that under physiological condition, AIP functions in stabilization of Aurora-A but not Aurora-B, though this regulation may be dependent on additional factors as well. Further, AIP siRNA induced cell cycle arrest at G2/M, which is consistent with anticipated loss of function of Aurora-A in these cells. Thus, our study provides the first evidence of a role for AIP in G2/M cell cycle progression by cooperatively regulating protein stabilization of its up-stream regulator, Aurora-A kinase through protein-protein interaction as well as protein phosphorylation.

Phosphorylated Cofilin Not Inactive After All

Cofilin is an actin-depolymerizing factor, and this activity is inhibited when the protein is phosphorylated by LIM-kinase or TES-kinase. In an attempt to identify the mechanism by which the muscarinic acetylcholine receptor activated phospholipase D (PLD) downstream of the Rho effector Rho-kinase, Han et al . found that in the fibroblast cell line HEK293 and the neuroblastoma cell line N1E-115, PLD1 was not directly phosphorylated by Rho-kinase, but instead carbachol-stimulated PLD1 activation was inhibited by expression of a kinase-deficient LIM-kinase 1, which is a target for Rho-kinase. However, PLD1 was not a substrate for LIM-kinase 1 either, suggesting another step in the path from the receptor to PLD1. As cofilin is a known substrate for LIM-kinase 1, the authors overexpressed cofilin or a nonphosphorylatable mutant S3A cofilin and showed that carbachol-mediated stimulation of PLD1 was enhanced in the presence of excess wild-type cofilin and inhibited by S3A cofilin. RNAi-mediated depletion of cofilin also reduced carbachol-mediated activation of PLD1, consistent with a role for endogenous cofilin. Expression of a phosphorylation-mimic mutant of cofilin S3D also enhanced carbachol-stimulated PLD1 activation, whereas overexpression of the cofilin phosphatase slingshot inhibited PLD activation. Pull-down assays, as well as colocalization assays and coimmunoprecipitation assays with transfected cells, showed a direct interaction between PLD1 and phosphorylated, but not unphosphorylated, cofilin. If the interaction between cofilin and PLD1 was disrupted by expressing a fragment of PLD1 (F-3 fragment) responsible for mediating the interaction, then carbachol-mediated PLD1 activation was inhibited. Carbachol stimulation of stress fiber formation was also inhibited by the F-3 fragment. Carbachol stimulated the transient phosphorylation of cofilin and increased the interaction between the cofilin and PLD1 in coimmunoprecipitation assays. Phosphorylated cofilin stimulated PLD1 activity in vitro. Thus, phosphorylated cofilin is not an inactive entity but rather interacts with partners other than actin, such as PLD1. L. Han, M. B. Stope, M. López de Jesús, P. A. Oude Weernink, M. Urban, T. Wieland, D. Rosskopf, K. Mizuno, K. H. Jakobs, M. Schmidt, Direct stimulation of receptor-controlled phospholipase D1 by phospho-cofilin. EMBO J. 26 , 4189-4202 (2007). [PubMed]

Direct stimulation of receptor-controlled phospholipase D1 by phospho-cofilin

The activity state of cofilin, which controls actin dynamics, is driven by a phosphorylation-dephosphorylation cycle. Phosphorylation of cofilin by LIM-kinases results in its inactivation, a process supported by 14-3-3zeta and reversed by dephosphorylation by slingshot phosphatases. Here we report on a novel cellular function for the phosphorylation-dephosphorylation cycle of cofilin. We demonstrate that muscarinic receptor-mediated stimulation of phospholipase D1 (PLD1) is controlled by LIM-kinase, slingshot phosphatase as well as 14-3-3zeta, and requires phosphorylatable cofilin. Cofilin directly and specifically interacts with PLD1 and upon phosphorylation by LIM-kinase1, stimulates PLD1 activity, an effect mimicked by phosphorylation-mimic cofilin mutants. The interaction of cofilin with PLD1 is under receptor control and encompasses a PLD1-specific fragment (aa 585-712). Expression of this fragment suppresses receptor-induced cofilin-PLD1 interaction as well as PLD stimulation and actin stress fiber formation. These data indicate that till now designated inactive phospho-cofilin exhibits an active cellular function, and suggest that phospho-cofilin by its stimulatory effect on PLD1 may control a large variety of cellular functions.

IKKβ Promotes Tumorigenesis

The tuberous sclerosis complex composed of TSC1 and TSC2, both tumor suppressors, inhibits the activity of the mTOR (mammalian target of rapamycin) pathway, which controls protein synthesis, cell proliferation, and production of vascular endothelial growth factor (VEGF). Lee et al . report that inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β, stimulate the mTOR pathway (detected as phosphorylation of the mTOR substrate S6K) in breast cancer cell lines and untransformed mammary epithelial cells. Activation of the mTOR pathway by TNF-α was lost if activity of inhibitor of nuclear factor κB kinase β (IKKβ) was decreased pharmacologically or by transfection of a dominant-negative inhibitor, by siRNA-mediated knockdown, or in Ikkβ knockout cells. IKKβ was not required for stimulation of mTOR signaling by growth factors or mitogens. IKKβ directly interacted with TSC1 and phosphorylated TSC1 on Ser 487 and Ser 511 both in vitro and in vivo. In cells expressing a mutant TSC1 [TSC1(SSAA)] in which the two serines were replaced with alanines, TNF-α failed to stimulate mTOR activity. Coimmunoprecipitation experiments with cells exposed to TNF-α suggested that phosphorylation by IKKβ disrupted the formation of the TSC1 and TSC2 complex, which is required for this complex to inhibit mTOR activity. In vivo tumorigenesis assays using TSC1 or TSC1(SSAA) or a phosphorylation mimic mutant [TSC1(SSDD)] showed that only wild-type TSC1 and TSC1(SSAA) had tumor-suppressing effects and that the blood vessel density of tumors with TSC1(SSDD) was higher than in the tumors with wild-type or TSC1(SSAA). Wild-type and TSC1(SSAA), but not TSC1(SSDD), exhibited the antiangiogenic effect of decreasing VEGF secretion when transfected into cultured cells. Thus, IKKβ-mediated inactivation of TSC1 may be one mechanism by which inflammation contributes to cancer progression. Phosphorylation of TSC1 at Ser 511 correlated with breast cancer tumors that were also high in phosphorylated (and active) IKKβ and high in TNF-α. Additionally, breast tumor samples with high phosphorylated TSC1 (Ser 511 ) and high phosphorylated S6K were associated with poor survival. Thus, this inflammatory connection appears to be an important aspect to cancer prognosis. D.-F. Lee, H.-P. Kuo, C.-T. Chen, J.-M. Hsu, C.-K. Chou, Y. Wei, H.-L. Sun, L.-Y. Li, B. Ping, W.-C. Huang, X. He, J.-Y. Hung, C.-C. Lai, Q. Ding, J.-L. Su, J.-Y. Yang, A. A. Sahin, G. N. Hortobagyi, F.-J. Tsai, C.-H. Tsai, M.-C. Hung, IKKβ suppression of TSC1 links inflammation and tumor angiogenesis via the mTOR pathway. Cell 130 , 440-455 (2007). [Online Journal]