Intracellular signalling: PDK1--a kinase at the hub of things.
(9/128527)
Phosphoinositide-dependent kinase 1 (PDK1) is at the hub of many signalling pathways, activating PKB and PKC isoenzymes, as well as p70 S6 kinase and perhaps PKA. PDK1 action is determined by colocalization with substrate and by target site availability, features that may enable it to operate in both resting and stimulated cells. (+info)
Alzheimer's disease: clues from flies and worms.
(10/128527)
Presenilin mutations give rise to familial Alzheimer's disease and result in elevated production of amyloid beta peptide. Recent evidence that presenilins act in developmental signalling pathways may be the key to understanding how senile plaques, neurofibrillary tangles and apoptosis are all biochemically linked. (+info)
Transformation mediated by RhoA requires activity of ROCK kinases.
(11/128527)
BACKGROUND: The Ras-related GTPase RhoA controls signalling processes required for cytoskeletal reorganisation, transcriptional regulation, and transformation. The ability of RhoA mutants to transform cells correlates not with transcription but with their ability to bind ROCK-I, an effector kinase involved in cytoskeletal reorganisation. We used a recently developed specific ROCK inhibitor, Y-27632, and ROCK truncation mutants to investigate the role of ROCK kinases in transcriptional activation and transformation. RESULTS: In NIH3T3 cells, Y-27632 did not prevent the activation of serum response factor, transcription of c-fos or cell cycle re-entry following serum stimulation. Repeated treatment of NIH3T3 cells with Y-27632, however, substantially disrupted their actin fibre network but did not affect their growth rate. Y-27632 blocked focus formation by RhoA and its guanine-nucleotide exchange factors Dbl and mNET1. It did not affect the growth rate of cells transformed by Dbl and mNET1, but restored normal growth control at confluence and prevented their growth in soft agar. Y-27632 also significantly inhibited focus formation by Ras, but had no effect on the establishment or maintenance of transformation by Src. Furthermore, it significantly inhibited anchorage-independent growth of two out of four colorectal tumour cell lines. Consistent with these data, a truncated ROCK derivative exhibited weak ability to cooperate with activated Raf in focus formation assays. CONCLUSIONS: ROCK signalling is required for both the establishment and maintenance of transformation by constitutive activation of RhoA, and contributes to the Ras-transformed phenotype. These observations provide a potential explanation for the requirement for Rho in Ras-mediated transformation. Moreover, the inhibition of ROCK kinases may be of therapeutic use. (+info)
Concomitant activation of pathways downstream of Grb2 and PI 3-kinase is required for MET-mediated metastasis.
(12/128527)
The Met tyrosine kinase - the HGF receptor - induces cell transformation and metastasis when constitutively activated. Met signaling is mediated by phosphorylation of two carboxy-terminal tyrosines which act as docking sites for a number of SH2-containing molecules. These include Grb2 and p85 which couple the receptor, respectively, with Ras and PI 3-kinase. We previously showed that a Met mutant designed to obtain preferential coupling with Grb2 (Met2xGrb2) is permissive for motility, increases transformation, but - surprisingly - is impaired in causing invasion and metastasis. In this work we used Met mutants optimized for binding either p85 alone (Met2xPI3K) or p85 and Grb2 (MetPI3K/Grb2) to evaluate the relative importance of Ras and PI 3-kinase as downstream effectors of Met. Met2xPI3K was competent in eliciting motility, but not transformation, invasion, or metastasis. Conversely, MetP13K/Grb2 induced motility, transformation, invasion and metastasis as efficiently as wild type Met. Furthermore, the expression of constitutively active PI 3-kinase in cells transformed by the Met2xGrb2 mutant, fully rescued their ability to invade and metastasize. These data point to a central role for PI 3-kinase in Met-mediated invasiveness, and indicate that simultaneous activation of Ras and PI 3-kinase is required to unleash the Met metastatic potential. (+info)
Tyrosine phosphorylation and complex formation of Cbl-b upon T cell receptor stimulation.
(13/128527)
Cbl-b, a mammalian homolog of Cbl, consists of an N-terminal region (Cbl-b-N) highly homologous to oncogenic v-Cbl, a Ring finger, and a C-terminal region containing multiple proline-rich stretches and potential tyrosine phosphorylation sites. In the present study, we demonstrate that upon engagement of the T cell receptor (TCR), endogenous Cbl-b becomes rapidly tyrosine-phosphorylated. In heterogeneous COS-1 cells, Cbl-b was phosphorylated on tyrosine residues by both Syk- (Syk/Zap-70) and Src- (Fyn/Lck) family kinases, with Syk kinase inducing the most prominent effect. Syk associates and phosphorylates Cbl-b in Jurkat T cells. A Tyr-316 Cbl-binding site in Syk was required for the association with and for the maximal tyrosine phosphorylation of Cbl-b. Mutation at a loss-of-function site (Gly-298) in Cbl-b-N disrupts its interaction with Syk. Cbl-b constitutively binds Grb2 and becomes associated with Crk-L upon TCR stimulation. The Grb2- and the Crk-L-binding regions were mapped to the C-terminus of Cbl-b. The Crk-L-binding sites were further determined to be Y655DVP and Y709KIP, with the latter being the primary binding site. Taken together, these results implicate that Cbl-b is involved in TCR-mediated intracellular signaling pathways. (+info)
Polarized distribution of Bcr-Abl in migrating myeloid cells and co-localization of Bcr-Abl and its target proteins.
(14/128527)
Bcr-Abl plays a critical role in the pathogenesis of Philadelphia chromosome-positive leukemia. Although a large number of substrates and interacting proteins of Bcr-Abl have been identified, it remains unclear whether Bcr-Abl assembles multi-protein complexes and if it does where these complexes are within cells. We have investigated the localization of Bcr-Abl in 32D myeloid cells attached to the extracellular matrix. We have found that Bcr-Abl displays a polarized distribution, colocalizing with a subset of filamentous actin at trailing portions of migrating 32D cells, and localizes on the cortical F-actin and on vesicle-like structures in resting 32D cells. Deletion of the actin binding domain of Bcr-Abl (Bcr-AbI-AD) dramatically enhances the localization of Bcr-Abl on the vesicle-like structures. These distinct localization patterns of Bcr-Abl and Bcr-Abl-AD enabled us to examine the localization of Bcr-Abl substrate and interacting proteins in relation to Bcr-Abl. We found that a subset of biochemically defined target proteins of Bcr-Abl redistributed and co-localized with Bcr-Abl on F-actin and on vesicle-like structures. The co-localization of signaling proteins with Bcr-Abl at its sites of localization supports the idea that Bcr-Abl forms a multi-protein signaling complex, while the polarized distribution and vesicle-like localization of Bcr-Abl may play a role in leukemogenesis. (+info)
Growth inhibition of breast cancer cells by Grb2 downregulation is correlated with inactivation of mitogen-activated protein kinase in EGFR, but not in ErbB2, cells.
(15/128527)
Increased breast cancer growth has been associated with increased expression of epidermal growth factor receptor (EGFR) and ErbB2 receptor tyrosine kinases (RTKs). Upon activation, RTKs may transmit their oncogenic signals by binding to the growth factor receptor bound protein-2 (Grb2), which in turn binds to SOS and activates the Ras/Raf/MEK/mitogen-activated protein (MAP) kinase pathway. Grb2 is important for the transformation of fibroblasts by EGFR and ErbB2; however, whether Grb2 is also important for the proliferation of breast cancer cells expressing these RTKs is unclear. We have used liposomes to deliver nuclease-resistant antisense oligodeoxynucleotides (oligos) specific for the GRB2 mRNA to breast cancer cells. Grb2 protein downregulation could inhibit breast cancer cell growth; the degree of growth inhibition was dependent upon the activation and/or endogenous levels of the RTKs. Grb2 inhibition led to MAP kinase inactivation in EGFR, but not in ErbB2, breast cancer cells, suggesting that different pathways might be used by EGFR and ErbB2 to regulate breast cancer growth. (+info)
The MAP kinase ERK2 inhibits the cyclic AMP-specific phosphodiesterase HSPDE4D3 by phosphorylating it at Ser579.
(16/128527)
The extracellular receptor stimulated kinase ERK2 (p42(MAPK))-phosphorylated human cAMP-specific phosphodiesterase PDE4D3 at Ser579 and profoundly reduced ( approximately 75%) its activity. These effects could be reversed by the action of protein phosphatase PP1. The inhibitory state of PDE4D3, engendered by ERK2 phosphorylation, was mimicked by the Ser579-->Asp mutant form of PDE4D3. In COS1 cells transfected to express PDE4D3, challenge with epidermal growth factor (EGF) caused the phosphorylation and inhibition of PDE4D3. This effect was blocked by the MEK inhibitor PD98059 and was not apparent using the Ser579-->Ala mutant form of PDE4D3. Challenge of HEK293 and F442A cells with EGF led to the PD98059-ablatable inhibition of endogenous PDE4D3 and PDE4D5 activities. EGF challenge of COS1 cells transfected to express PDE4D3 increased cAMP levels through a process ablated by PD98059. The activity of the Ser579-->Asp mutant form of PDE4D3 was increased by PKA phosphorylation. The transient form of the EGF-induced inhibition of PDE4D3 is thus suggested to be due to feedback regulation by PKA causing the ablation of the ERK2-induced inhibition of PDE4D3. We identify a novel means of cross-talk between the cAMP and ERK signalling pathways whereby cell stimuli that lead to ERK2 activation may modulate cAMP signalling. (+info)