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A S Alberts

Publications and source records attributed to A S Alberts.

At least 19 recordsLinked to original sources
PubMed2001

Cdc42, dynein, and dynactin regulate MTOC reorientation independent of Rho-regulated microtubule stabilization.

In migrating adherent cells such as fibroblasts and endothelial cells, the microtubule-organizing center (MTOC) reorients toward the leading edge [1-3]. MTOC reorientation repositions the Golgi toward the front of the cell [1] and contributes to directional migration [4]. The mechanism of MTOC reorientation and its relation to the formation of stabilized microtubules (MTs) in the leading edge, which occurs concomitantly with MTOC reorientation [3], is unknown. We show that serum and the serum lipid, lysophosphatidic acid (LPA), increased Cdc42 GTP levels and triggered MTOC reorientation in serum-starved wounded monolayers of 3T3 fibroblasts. Cdc42, but not Rho or Rac, was both sufficient and necessary for LPA-stimulated MTOC reorientation. MTOC reorientation was independent of Cdc42-induced changes in actin and was not blocked by cytochalasin D. Inhibition of dynein or dynactin blocked LPA- and Cdc42-stimulated MTOC reorientation. LPA also stimulates a Rho/mDia pathway that selectively stabilizes MTs in the leading edge [5, 6]; however, activators and inhibitors of MTOC reorientation and MT stabilization showed that each response was regulated independently. These results establish an LPA/Cdc42 signaling pathway that regulates MTOC reorientation in a dynein-dependent manner. MTOC reorientation and MT stabilization both act to polarize the MT array in migrating cells, yet these processes act independently and are regulated by separate Rho family GTPase-signaling pathways.

3T3 Cells↗
PubMed2001

mDia mediates Rho-regulated formation and orientation of stable microtubules.

Rho-GTPase stabilizes microtubules that are oriented towards the leading edge in serum-starved 3T3 fibroblasts through an unknown mechanism. We used a Rho-effector domain screen to identify mDia as a downstream Rho effector involved in microtubule stabilization. Constitutively active mDia or activation of endogenous mDia with the mDia-autoinhibitory domain stimulated the formation of stable microtubules that were capped and oriented towards the wound edge. mDia co-localized with stable microtubules when overexpressed and associated with microtubules in vitro. Rho kinase was not necessary for the formation of stable microtubules. Our results show that mDia is sufficient to generate and orient stable microtubules, and indicate that Dia-related formins are part of a conserved pathway that regulates the dynamics of microtubule ends.

3T3 Cells↗
PubMed2000

Identification of a carboxyl-terminal diaphanous-related formin homology protein autoregulatory domain.

Mammalian and fungal Diaphanous-related formin homology (DRF) proteins contain several regions of conserved sequence homology. These include an amino-terminal GTPase binding domain (GBD) that interacts with activated Rho family members and formin homology domains that mediate targeting or interactions with signaling kinases and actin-binding proteins. DRFs also contain a conserved Dia-autoregulatory domain (DAD) in their carboxyl termini that binds the GBD. The GBD is a bifunctional autoinhibitory domain that is regulated by activated Rho. Expression of the isolated DAD in cells causes actin fiber formation and stimulates serum response factor-regulated gene expression. Inhibitor experiments show that the effects of exogenous DAD expression are dependent upon cellular Dia proteins. Alanine substitution of DAD consensus residues that disrupt GBD binding also eliminate DAD biological activity. Thus, DAD expression activates nuclear signaling and actin remodeling by mimicking activated Rho and unlatching the autoinhibited state of the cellular complement of Dia proteins.

3T3 Cells↗
PubMed2000

Diaphanous-related formins bridge Rho GTPase and Src tyrosine kinase signaling.

We have examined the role of the mouse Diaphanous-related formin (DRF) Rho GTPase binding proteins, mDia1 and mDia2, in cell regulation. The DRFs are required for cytokinesis, stress fiber formation, and transcriptional activation of the serum response factor (SRF). 'Activated' mDia1 and mDia2 variants, lacking their GTPase binding domains, cooperated with Rho-kinase or ROCK to form stress fibers but independently activated SRF. Src tyrosine kinase associated and co-localized with the DRFs in endosomes and in mid-bodies of dividing cells. Inhibition of Src also blocked cytokinesis, SRF induction by activated DRFs, and cooperative stress fiber formation with active ROCK. Our results show that the DRF proteins couple Rho and Src during signaling and the regulation of actin dynamics.

3T3 Cells↗
PubMed2000

p21-activated kinase 1 plays a critical role in cellular activation by Nef.

The activation of Nef-associated kinase (NAK) by Nef from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. This induction occurs via the guanine nucleotide exchange factor Vav and the small GTPases Rac1 and Cdc42. In this study, we identified NAK as p21-activated kinase 1 (PAK1). PAK1 bound to Nef in vitro and in vivo. Moreover, the induction of cytoskeletal rearrangements such as the formation of trichopodia, the activation of Jun N-terminal kinase, and the increase of viral production were blocked by an inhibitory peptide that targets the kinase activity of PAK1 (PAK1 83-149). These results identify NAK as PAK1 and emphasize the central role its kinase activity plays in cytoskeletal rearrangements and cellular signaling by Nef.

Animals↗
PubMed1999

Activation of Vav by Nef induces cytoskeletal rearrangements and downstream effector functions.

Nef of primate lentiviruses is critical for high levels of viremia and the progression to AIDS. Nef associates with and activates a serine/threonine kinase (Nef-associated kinase [NAK]) via the small GTPases Rac1 and Cdc42. We identified the protooncogene and guanine nucleotide exchange factor Vav as the specific binding partner of Nef proteins from HIV-1 and SIV. The interaction between Nef and Vav led to increased activity of Vav and its downstream effectors. Both cytoskeletal changes and the activation of c-Jun N-terminal kinase (JNK) were observed. Furthermore, a dominant-negative Vav protein inhibited NAK activation and viral replication. Thus, the interaction between Nef and Vav initiates a signaling cascade that changes structural and physiological parameters in the infected cell.

Amino Acid Sequence↗
PubMed1998

Activation of RhoA and SAPK/JNK signalling pathways by the RhoA-specific exchange factor mNET1.

We have characterized the DH domain protein mNET1, a Rho-family guanine nucleotide exchange factor (GEF). N-terminal truncation of mNET1 generates an activated transforming form of the protein, mNET1DeltaN, which acts as a GEF for RhoA but not Cdc42 or Rac1. In NIH 3T3 cells, activated mNET1 induces formation of actin stress fibres and potentiates activity of the transcription factor serum response factor. Inhibitor studies show that these processes are dependent on RhoA and independent of Cdc42 or Rac1. In contrast to the GTPase-deficient RhoA.V14 mutant, however, expression of activated mNET1 also activates the SAPK/JNK pathway. This requires mNET1 GEF activity, since it is blocked by point mutations in the mNET1 DH domain and its C-terminal pleckstrin homology (PH) domain, and by the dominant-interfering RhoA mutant RhoA.N19. Although mNET1DeltaN-induced SAPK/JNK activation requires a C3 transferase-sensitive GTPase, it occurs independently of the generation of titratable GTP-bound RhoA. Thus, mNET1 can activate signalling pathways in addition to those directly controlled by activated RhoA.

3T3 Cells↗
PubMed1998

Analysis of RhoA-binding proteins reveals an interaction domain conserved in heterotrimeric G protein beta subunits and the yeast response regulator protein Skn7.

To identify potential RhoA effector proteins, we conducted a two-hybrid screen for cDNAs encoding proteins that interact with a Gal4-RhoA.V14 fusion protein. In addition to the RhoA effector ROCK-I we identified cDNAs encoding Kinectin, mDia2 (a p140 mDia-related protein), and the guanine nucleotide exchange factor, mNET1. ROCK-I, Kinectin, and mDia2 can bind the wild type forms of both RhoA and Cdc42 in a GTP-dependent manner in vitro. Comparison of the ROCK-I and Kinectin sequences revealed a short region of sequence homology that is both required for interaction in the two-hybrid assay and sufficient for weak interaction in vitro. Sequences related to the ROCK-I/Kinectin sequence homology are present in heterotrimeric G protein beta subunits and in the Saccharomyces cerevisiae Skn7 protein. We show that beta2 and Skn7 can interact with mammalian RhoA and Cdc42 and yeast Rho1, both in vivo and in vitro. Functional assays in yeast suggest that the Skn7 ROCK-I/Kinectin homology region is required for its function in vivo.

Amino Acid Sequence↗
PubMed1998

RhoA effector mutants reveal distinct effector pathways for cytoskeletal reorganization, SRF activation and transformation.

The RhoA GTPase regulates diverse cellular processes including cytoskeletal reorganization, transcription and transformation. Although many different potential RhoA effectors have been identified, including two families of protein kinases, their roles in RhoA-regulated events remain unclear. We used a genetic screen to identify mutations at positions 37-42 in the RhoA effector loop that selectively disrupt effector binding, and used these to investigate the role of RhoA effectors in the formation of actin stress fibres, activation of transcription by serum response factor (SRF) and transformation. Interaction with the ROCK kinase and at least one other unidentified effector is required for stress fibre formation. Signalling to SRF by RhoA can occur in the absence of RhoA-induced cytoskeletal changes, and did not correlate with binding to any of the effectors tested, indicating that it may be mediated by an unknown effector. Binding to ROCK-I, but not activation of SRF, correlated with the activity of RhoA in transformation. The effector mutants should provide novel approaches for the functional study of RhoA and isolation of effector molecules involved in specific signalling processes.

3T3 Cells↗
PubMed1998

Activation of SRF-regulated chromosomal templates by Rho-family GTPases requires a signal that also induces H4 hyperacetylation.

Constitutively active forms of the small GTPases RhoA (RhoA.V14) and Cdc42 (Cdc42.V12) induce expression of extrachromosomal SRF reporter genes in microinjection experiments, but only Cdc42.V12 can efficiently activate a chromosomal template. Both SAPK/JNK-dependent or -independent signals can cooperate with RhoA.V14 to activate chromosomal SRF reporters, and it is SAPK/JNK activation by Cdc42.V12 that allows it to activate chromosomal templates. Cooperating signals can be bypassed by deacetylase inhibitors. Three findings show that histone H4 hyperacetylation is one target for cooperating signals, although it alone is not sufficient: (1) Cdc42.V12, but not RhoA.V14, induces H4 hyperacetylation; (2) cooperating signals use the same SAPK/JNK-dependent or -independent pathways to induce H4 hyperacetylation; (3) growth factor and stress stimuli induce substantial H4 hyperacetylation, detectable in reporter gene chromatin. These data establish a link between signal-regulated acetylation events and gene transcription.

3T3 Cells↗
PubMed1998

Quantifying the radiation dosage to individual skeletal lesions treated with samarium-153-EDTMP.

UNLABELLED: Samarium-153ethylenediaminetetramethylenephosphonate (EDTMP) is used in the treatment of painful skeletal lesions. This study attempted to quantify the radiation dosage to individual lesions on both the macroscopic and microscopic level. METHODS: A gamma camera-based quantification technique was adapted and refined for 153Sm. The accuracy of the technique was determined by using a realistic phantom. The activity and volume of lesions as well as normal bone were determined and used to estimate the radiation dosages to these regions. Two patients died of unrelated causes shortly after receiving 153Sm-EDTMP. This made it possible to compare the gamma camera results with direct measurements. It also allowed for autoradiographic examination of the lesions. Finally, the microscopic radiation dosages were estimated. RESULTS: The phantom study indicated that the quantification technique was off, on average, by 4.1% (s.d. = 8.1%). The absolute activity concentration of trabecular bone was found to be approximately 0.22 MBq/g, and that of cortical bone was found to be approximately 0.1 MBq/g, regardless of the dosage administered. The corresponding concentrations for lesions were between 3 and 7 times higher than that of normal bone, with no apparent ceiling. From these results, the macroscopic radiation dosage could be estimated. The dosage to normal bone varied between 0.9 and 3.9 cGy x kg/MBq, and that of the lesions varied between 5.2 and 27.1 cGy x kg/MBq. The autopsy results confirmed that the gamma camera technique was accurate. The autoradiography showed clearly that the activity was associated with the surface of the bone. From these findings, the microscopic radiation dosage distribution was estimated for cortical and trabecular bone as well as osteoblastic lesions. The variation in the microscopic dosage compared to the macroscopic dosage was quite large. Microscopic dosages, when compared to the macroscopic dosages, were as high as 965% and as low as 14.9%. CONCLUSION: The techniques used have been proven to be accurate. The activity in normal bone may be at a ceiling value for all the administered doses, which could explain the small variation. This is not true for the lesions. The large variation in dosages on a microscopic scale, combined with the ceiling in normal bone, may explain the lower than expected toxicity and relatively quick relapse of the patients.

Autoradiography↗
PubMed1997

Dose response relationship and multiple dose efficacy and toxicity of samarium-153-EDTMP in metastatic cancer to bone.

INTRODUCTION: The optimal dose of samarium-153-EDTMP (153Sm-EDTMP) for effective palliation of painful metastases to bone is under investigation. It is not known whether increased doses of 153Sm EDTMP will lead to better and longer pain and tumour control and survival. Multiple dose efficacy and toxicity is of importance as most Patients will require prolonged support for pain. METHODS: Twenty-eight (28) patients were treated with 0.75 mCi/kg, 35 patients with 1.5 mCi/kg and 19 patients with 3 mCi/kg in three sequential Phase I-II trials. Multiple doses were given to patients on the 0.75 mCi/kg and 1.5 mCi/kg dose levels. RESULTS: At all dose levels adequate pain control was achieved in 78-95% of patients. The duration of pain control was 40-56 days with the best results in the 1.5 mCi/kg group (56 days). There is no evidence that increasing dose leads to better and longer pain control, tumour response and survival, but toxicity is increased. Multiple doses can be given with acceptable toxicity and pain control, however, only 38% of patients will qualify for multiple treatments. CONCLUSION: 153Sm-EDTMP provides adequate and safe palliation but multiple doses can only be given in 38% of patients. There is not a clear dose-response relationship. The length of pain control is satisfactory but not ideal and hospitalisation for 4 days every 6-8 weeks is a disadvantage. Further research is required to combine 153Sm-EDTMP with cytostatics and to administer it on an out patient basis.

Bone Neoplasms↗
PubMed1996

Evaluation of samarium-153 and holmium-166-EDTMP in the normal baboon model.

Bone-seeking radiopharmaceuticals such as ethylenediaminetetramethylene phosphonate (EDTMP) complexes of samarium-153 and holmium-166 are receiving considerable attention for therapeutic treatment of bone metastases. In this study, using the baboon experimental model, multicompartmental analysis revealed that with regard to pharmacokinetics, biodistribution, and skeletal localisation, 166Ho-EDTMP was significantly inferior to 153Sm-EDTMP and 99mTc-MDP. A more suitable 166Ho-bone-seeking agent should thus be sought for closer similarity to 153Sm-EDTMP to exploit fully the therapeutic potential of its shorter half-life and more energetic beta radiation.

Absorption↗
PubMed1995

Samarium-153-EDTMP for palliation of ankylosing spondylitis, Paget's disease and rheumatoid arthritis.

Samarium-153-EDTMP is an effective agent for palliation of widespread skeletal metastases because it concentrates in bone metastases which have an osteoblastic component. Similar concentration in areas of osteoblastic activity in ankylosing spondylitis, Paget's disease and rheumatoid arthritis suggests a possible new treatment approach. Three patients with ankylosing spondylitis, one patient with Paget's disease and one patient with rheumatoid arthritis were treated with 153Sm-EDTMP. Objective and subjective improvement was noted, especially in ankylosing spondylitis patients. Samarium-153-EDTMP has disease-modifying potential in ankylosing spondylitis and Paget's disease and has palliative value in resistant rheumatoid arthritis. Further trials to determine optimal dose, treatment scheduling, long-term disease-modifying potential and toxicity are needed.

Arthritis, Rheumatoid↗
PubMed1995

Radiation of benign disease--a South African perspective.

Radiation of benign disease is controversial. The approach of 55 South African radiation oncologists to 14 benign conditions was obtained and compared to available American data. In general, the approach was conservative except in cases of keloids. The South African and American approaches are remarkably similar (except with regard to ankylosing spondylitis). Private radiation oncologists are less conservative than their hospital counterparts. Randomised trials are needed firmly to establish efficacy and long-term side-effects.

Aged↗
PubMed1994

Activation of cAMP and mitogen responsive genes relies on a common nuclear factor.

A number of signalling pathways stimulate transcription of target genes through nuclear factors whose activities are primarily regulated by phosphorylation. Cyclic AMP regulates the expression of numerous genes, for example, through the protein kinase-A (PKA)-mediated phosphorylation of transcription factor CREB at Ser 133. Although phosphorylation may stimulate transcriptional activators by modulating their nuclear transport or DNA-binding affinity, CREB belongs to a class of proteins whose phosphorylation appears specifically to enhance their trans-activation potential. Recent work describing a phospho-CREB binding protein (CBP) which interacts specifically with the CREB trans-activation domain prompted us to examine whether CBP is necessary for cAMP regulated transcription. We report here that microinjection of an anti-CBP antiserum into fibroblasts can inhibit transcription from a cAMP responsive promoter. Surprisingly, CBP also cooperates with upstream activators such as c-Jun, which are involved in mitogen responsive transcription. We propose that CBP is recruited to the promoter through interaction with certain phosphorylated factors, and that CBP may thus play a critical role in the transmission of inductive signals from cell surface receptor to the transcriptional apparatus.

3T3 Cells↗
PubMed1994

Recombinant cyclic AMP response element binding protein (CREB) phosphorylated on Ser-133 is transcriptionally active upon its introduction into fibroblast nuclei.

To date, it has not been possible to determine whether the single phosphorylation of the cyclic AMP response element binding factor (CREB) at Ser-133 is sufficient for the transcriptional activation by cAMP-mediated pathways. Previous in vivo studies investigating this point have relied upon transfection of cyclic AMP-dependent kinase (cAPK) or its activation by treatment of cells with cell-permeable cAMP analogs. However, as numerous cellular proteins, including CREB, are substrates for activated cAPK, the possibility remains that cAPK substrates other than CREB are required for the transcriptional activity of CRE-containing promoters. To further address this, we compared the activity of recombinant CREB phosphorylated on Ser-133 in both cell-free transcription assays and in vivo after introduction of the same preparations into fibroblasts by microinjection. The activity of phosphorylated CREB, nonphosphorylated CREB, and a mutant form of CREB, containing Ala substituted for Ser at position 133, was found to be nearly identical in cell-free in vitro transcription assays. In contrast, we found that only the phosphorylated CREB microinjected into fibroblasts resulted in the stimulation of expression of CRE-regulated genes. These results suggest that phosphorylation of CREB on Ser-133 directly stimulates its ability to transactivate gene expression in intact cells.

3T3 Cells↗