The GAP1 family of GTPase-activating proteins: spatial and temporal regulators of small GTPase signalling.

Ras proteins are binary switches that, by cycling between inactive GDP-bound and active GTP-bound conformations, regulate multiple cellular signalling pathways including those that control cell growth, differentiation and survival. Approximately 30% of all human tumours express Ras-containing oncogenic mutations that lock the protein into a constitutively active conformation. The activation status of Ras is regulated by two groups of proteins: GEFs (guanine nucleotide-exchange factors) bind to Ras and enhance the exchange of GDP for GTP, thereby activating it, whereas GAPs (GTPase-activating proteins) inactivate Ras by binding to the GTP-bound form and enhancing the hydrolysis of the bound nucleotide back to GDP. In this review, we focus on a group of key regulators of Ras inactivation, the GAP1 family of Ras-GAPs. The members of this family are GAP1m, GAP1IP4BP, CAPRI (Ca2+-promoted Ras inactivator) and RASAL (Ras-GTPase-activating-like protein) and, as we will discuss, they are emerging as important modulators of Ras and small GTPase signalling that are subject to regulation by a diverse array of events and second messenger signals.

Microtubule-associated proteins (MAPs) regulate cAMP signalling through exchange protein directly activated by cAMP (EPAC).

cAMP is an essential signalling molecule whose concentration in cells is regulated by a wide range of hormones. A large number of diseases, including cancer and asthma, are linked to improper regulation of the cAMP signalling system, and manipulation of cAMP levels by pharmaceutical agents has proven therapeutic benefit. The action of cAMP in cells is mediated through the signalling enzymes PKA (protein kinase A) and EPAC (exchange protein directly activated by cAMP). The study of the function of these proteins is essential to understand the role of cAMP in controlling disease. We have found that EPAC interacts with an ancillary protein, called LC2 (light chain 2), and this interaction enhances EPAC's ability to activate its substrate protein, Rap1 GTPase. This is an important finding because Rap1 is involved in the control of cell migration and cell shape, functions that are disrupted in diseases like cancer. LC2 appears to enhance EPAC activity towards Rap1 by increasing the ability of EPAC to interact with cAMP, so that EPAC activation occurs at lower concentrations of cAMP. The design of inhibitors that disrupt or enhance EPAC1-LC2 interaction may therefore form the basis of future therapeutics for diseases where cAMP signalling through Rap1 is improperly regulated.

Reduced delays in A&E for elderly patients with hip fractures.

Published guidelines recommend early transfer of patients with hip fractures to hospital wards and avoidance of unnecessary delays in A&E. We describe a protocol whereby the liaison of an orthopaedic trauma co-ordinator with A&E reduced A&E-to-ward transfer times by 43%. Following introduction of the new protocol, 39% of hip fracture patients were in a ward bed within 3 h of admission to A&E compared to 4% previously. The new protocol also reduces administrative workload for the on-call orthopaedic SHOs.

Factors delaying surgical treatment of hip fractures in elderly patients.

BACKGROUND: Published guidelines recommend early surgical treatment of hip fractures in elderly patients. Understanding the factors that delay surgical intervention is essential in order to introduce changes that will facilitate early treatment. AIM: To determine the factors delaying surgical treatment of hip fractures in elderly patients for more than 24 h. PATIENTS AND METHODS: Assessment of 163 consecutive patients undergoing surgery for hip fractures at the trauma unit of Manchester Royal Infirmary. RESULTS: Only 72/163 (44.2%) patients had their operation within 24 h of presenting to hospital. The remaining 91 patients had a total of 239 days delay (in excess of the initial 24 h) for surgical treatment. Active medical problems (56.5%) and a wait for medical investigations (19.7%) caused most delays. Lack of operating theatre time and Sunday trauma lists caused 23.8% of delays. CONCLUSIONS: Medical problems account for most delays of surgical treatment of hip fractures. A multidisciplinary approach, with early input by medical and anaesthetic teams, is essential in managing such patients. Established protocols may reduce waiting times for essential investigations.

The development of a protocol in using day surgery for minor orthopaedic trauma patients.

We examined all minor orthopaedic trauma patients requiring surgical intervention at Manchester Royal Infirmary over a 6-month period to determine whether minor trauma patients meet the day surgery requirements in terms of medical fitness, analgesia requirements and postoperative complications. A total of 83 patients had surgery for minor orthopaedic trauma in the defined period. Of these patients, 79 (95.2%) were medically fit, 45 (70%) were admitted to a hospital ward and 19 (30%) were brought back to day surgery. None of the patients attending day surgery developed postoperative complications or required return to hospital. The majority of patients admitted to a ward required simple or no analgesia pre- (95.5%) and postoperatively (100%), and most were discharged on the same (55.6%) or next (35.6%) day of their operation. We suggest a protocol whereby patients with minor trauma are brought back to day surgery. This could potentially reduce pressures on bed availability.

Extracellular matrix composition determines the transcriptional response to epidermal growth factor receptor activation.

The transcriptional response to epidermal growth factor (EGF) was examined in a cultured cell model of adhesion. Gene expression was monitored in human embryonic kidney cells (HEK293) after attachment of cells to the extracellular matrix (ECM) proteins, laminin, and fibronectin, by using complementary DNA microarrays printed with 1,718 individual human genes. Cluster analysis revealed that the influence of EGF on gene expression, either positive or negative, was largely independent of ECM composition. However, clusters of EGF-regulated genes were identified that were diagnostic of the type of ECM proteins to which cells were attached. In these clusters, attachment of cells to a laminin or fibronectin substrata specifically modified the direction of gene expression changes in response to EGF stimulation. For example, in HEK293 cells attached to fibronectin, EGF stimulated an increase in the expression of some genes; however, genes in the same group were nonresponsive or even suppressed in cells attached to laminin. Many of the genes regulated by EGF and ECM proteins in this manner are involved in ECM and cytoskeletal architecture, protein synthesis, and cell cycle control, indicating that cell responses to EGF stimulation can be dramatically affected by ECM composition.

Use of an activation-specific probe to show that Rap1A and Rap1B display different sensitivities to activation by forskolin in rat1 cells.

Rap1A and Rap1B are small GTPases of the Ras superfamily whose activation can be measured using a probe that interacts specifically with the GTP-bound forms of Rap1A and Rap1B. Using this procedure we demonstrate that the cyclic AMP-elevating agent forskolin activates both Rap1A and Rap1B in Rat1 cells. Whilst the protein kinase A inhibitor H89 ablated the ability of forskolin to cause cAMP response element binding protein (CREB) phosphorylation in Rat1 cells, it did not affect the ability of forskolin to activate either Rap1A and Rap1B. Forskolin differentially activated Rap1A and Rap1B isoforms in a time- and dose-dependent manner. The cAMP-specific type 4 family phosphodiesterase inhibitor rolipram potentiated the rate of activation of both Rap1A and Rap1B by forskolin challenge of Rat1 cells. Challenge of Rat1 cells with rolipram alone was able to elicit the phosphorylation of CREB but not activation of either Rap1A or Rap1B.

The RACK1 signaling scaffold protein selectively interacts with the cAMP-specific phosphodiesterase PDE4D5 isoform.

The WD-repeat protein receptor for activated C-kinase (RACK1) was identified by its interaction with the cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4D5 in a yeast two-hybrid screen. The interaction was confirmed by co-immunoprecipitation of native RACK1 and PDE4D5 from COS7, HEK293, 3T3-F442A, and SK-N-SH cell lines. The interaction was unaffected by stimulation of the cells with the phorbol ester phorbol 2-myristate 3-acetate. PDE4D5 did not interact with two other WD-repeat proteins, beta'-coatomer protein and Gsbeta, in two-hybrid tests. RACK1 did not interact with other PDE4D isoforms or with known PDE4A, PDE4B, and PDE4C isoforms. PDE4D5 and RACK1 interacted with high affinity (Ka approximately 7 nM) [corrected] when they were expressed and purified from Escherichia coli, demonstrating that the interaction does not require intermediate proteins. The binding of the E. coli-expressed proteins did not alter the kinetics of cAMP hydrolysis by PDE4D5 but caused a 3-4-fold change in its sensitivity to inhibition by the PDE4 selective inhibitor rolipram. The subcellular distributions of RACK1 and PDE4D5 were extremely similar, with the major amount of both proteins (70%) in the high speed supernatant (S2) fraction. Analysis of constructs with specific deletions or single amino acid mutations in PDE4D5 demonstrated that a small cluster of amino acids in the unique amino-terminal region of PDE4D5 was necessary for its interaction with RACK1. We suggest that RACK1 may act as a scaffold protein to recruit PDE4D5 and other proteins into a signaling complex.

Association with the SRC family tyrosyl kinase LYN triggers a conformational change in the catalytic region of human cAMP-specific phosphodiesterase HSPDE4A4B. Consequences for rolipram inhibition.

The cAMP-specific phosphodiesterase (PDE) HSPDE 4A4B(pde46) selectively bound SH3 domains of SRC family tyrosyl kinases. Such an interaction profoundly changed the inhibition of PDE4 activity caused by the PDE4-selective inhibitor rolipram and mimicked the enhanced rolipram inhibition seen for particulate, compared with cytosolic pde46 expressed in COS7 cells. Particulate pde46 co-localized with LYN kinase in COS7 cells. The unique N-terminal and LR2 regions of pde46 contained the sites for SH3 binding. Altered rolipram inhibition was triggered by SH3 domain interaction with the LR2 region. Purified LYN SH3 and human PDE4A LR2 could be co-immunoprecipitated, indicating a direct interaction. Protein kinase A-phosphorylated pde46 remained able to bind LYN SH3. pde46 was found to be associated with SRC kinase in the cytosol of COS1 cells, leading to aberrant kinetics of rolipram inhibition. It is suggested that pde46 may be associated with SRC family tyrosyl kinases in intact cells and that the ensuing SH3 domain interaction with the LR2 region of pde46 alters the conformation of the PDE catalytic unit, as detected by altered rolipram inhibition. Interaction between pde46 and SRC family tyrosyl kinases highlights a potentially novel regulatory system and point of signaling system cross-talk.

Growth hormone-dependent differentiation of 3T3-F442A preadipocytes requires Janus kinase/signal transducer and activator of transcription but not mitogen-activated protein kinase or p70 S6 kinase signaling.

The signals mediating growth hormone (GH)-dependent differentiation of 3T3-F442A preadipocytes under serum-free conditions have been studied. GH priming of cells was required before the induction of terminal differentiation by a combination of epidermal growth factor, tri-iodothyronine, and insulin. Cellular depletion of Janus kinase-2 (JAK-2) using antisense oligodeoxynucleotides (ODNs) prevented GH-stimulated JAK-2 and signal transducer and activator of transcription (STAT)-5 tyrosine phosphorylation and severely attenuated the ability of GH to promote differentiation. Although p42(MAPK)/p44(MAPK) mitogen-activated protein kinases were activated during GH priming, treatment of cells with PD 098059, which prevented activation of these kinases, did not block GH priming. However, antisense ODN-mediated depletion of mitogen-activated protein kinases from the cells showed that their expression was necessary for terminal differentiation. Similarly, although p70(s6k) was activated during GH priming, pretreatment of cells with rapamycin, which prevented the activation of p70(s6k), had no effect on GH priming. However, rapamycin did partially block epidermal growth factor, tri-iodothyronine, and insulin-stimulated terminal differentiation. By contrast, cellular depletion of STAT-5 with antisense ODNs completely abolished the ability of GH to promote differentiation. These results indicate that JAK-2, acting specifically via STAT-5, is necessary for GH-dependent differentiation of 3T3-F442A preadipocytes. Activation of p42(MAPK)/p44(MAPK) and p70(s6k) is not essential for the promotion of differentiation by GH, although these signals are required for GH-independent terminal differentiation.

The MAP kinase ERK2 inhibits the cyclic AMP-specific phosphodiesterase HSPDE4D3 by phosphorylating it at Ser579.

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.

Cyclic AMP potentiates growth hormone-dependent differentiation of 3T3-F442A preadipocytes: possible involvement of the transcription factor CREB.

We have examined the effects of cyclic AMP on the differentiation of 3T3-F442A preadipocytes. High concentrations of intracellular cyclic AMP potently inhibited differentiation whereas low concentrations of intracellular cyclic AMP, induced by a number of different agents, promoted differentiation. To analyse these effects of cyclic AMP more closely, we developed a two-phase protocol for the differentiation of 3T3-F442A cells. Growth hormone (GH) was necessary to prime confluent cells during the first phase, following which, the addition of insulin and other adipogenic agents then promoted terminal differentiation. Cyclic AMP potentiated the priming action of GH but exerted an inhibitory effect on terminal differentiation when added to cells which had previously been primed with GH showing that the effects of cyclic AMP on preadipocyte differentiation are stage-dependent. We analysed the stimulatory effects of cyclic AMP during GH priming and found that cyclic AMP induced phosphorylation of the cyclic AMP response element (CRE) binding protein CREB and activated transcription of a CRE-linked reporter gene. Furthermore, GH also stimulated CREB phosphorylation and activation and this effect was potentiated by cyclic AMP. These results suggest a mechanism for the synergistic priming of preadipocytes for terminal differentiation by cyclic AMP and GH via the activation of differentiation genes containing CREs.

Stimulation of p70S6 kinase via a growth hormone-controlled phosphatidylinositol 3-kinase pathway leads to the activation of a PDE4A cyclic AMP-specific phosphodiesterase in 3T3-F442A preadipocytes.

The challenge of 3T3-F442A fibroblasts with growth hormone led to both a decrease in the mobility on SDS/PAGE and activation of the PDE4A cyclic AMP-specific phosphodiesterase isoform PDE4A5. Activation was mediated by a JAK-2-dependent pathway coupled to the activation of phosphatidylinositol 3-kinase and p70S6 kinase. Activation was not dependent on the ability of growth hormone to stimulate ERK2 or protein kinase C or any effect on transcription. Blockade of activation of murine PDE4A5 ablated the ability of growth hormone to decrease intracellular cAMP levels. Antisense depletion of murine PDE4A5 mimicked the ability of rolipram to enhance the growth hormone-stimulated differentiation of 3T3-F442A cells to adipocytes. It is suggested that activation of PDE4A5 by growth hormone serves as a brake on the differentiation processes.

Intracellular compartmentalization of PDE4 cyclic AMP-specific phosphodiesterases.

The PDE4 cyclic AMP-specific phosphodiesterase family comprises a large number of different isoforms encoded by four distinct genes, with additional complexity arising through alternate mRNA splicing. This generates a number of distinct PDE4 isoforms with unique N-terminal regions. The range of such splice variants emanating from the four PDE4 genes appears to be highly conserved across species. One key role for such regions appears to be their potential to target isoforms to specific intracellular sites. Evidence for such a targeting role for these N-terminal regions can be gleaned by a variety of techniques. These include subcellular fractionation, confocal microscopy, binding assays to show association with proteins having src homology 3 (SH3) domains, and generation of chimeric constructs of these N-terminal regions with proteins that are normally expressed in the cytosol.

Cyclic AMP stimulates the phosphorylation and activation of p42 and p44 mitogen-activated protein kinases in 3T3-F442A preadipocytes.

The effects of cyclic AMP on mitogen-activated protein (MAP) kinase activation were investigated in 3T3-F442A preadipocytes. Several agents, which raise intracellular cyclic AMP levels by distinct mechanisms, induced a transient activation of the p42 and p44 isoforms of MAP kinase and of a MAP kinase kinase. Activation of MAP kinase by cyclic AMP was prevented by two distinct inhibitors of cyclic AMP-dependent protein kinase and by PD 098059, a specific inhibitor of the activation of the MAP kinase kinase MEK 1. Therefore, in contrast to most cell types studied, cyclic AMP exerts a positive influence on the MAP kinase pathway in 3T3-F442A preadipocytes at a level upstream of the activation of MAP kinase kinase.