Anticoagulation management during cross-clamping and bypass.

Anticoagulation is required for successful implementation of cardiopulmonary bypass (CPB), as well as for surgeries requiring temporary aortic occlusion. It is well established that both coagulation and fibrinolysis are activated during CPB (Teufelsbauer et al., 1992) [1]. Appropriate dosing, monitoring, and maintenance of anticoagulation are essential to prevent devastating thrombosis of the CPB circuit or the occluded aorta and to minimize the activation of the hemostatic system. Although numerous novel anticoagulants have been developed over the past decade, unfractionated heparin remains the primary anticoagulant utilized during these types of procedures, with monitoring systems primarily based upon the activated clotting time and/or heparin concentration. This article will review the current state of anticoagulation management during cross-clamp and CPB.

Intensive dietary intervention by a dietitian as a case manager among community dwelling older adults: the EDIT study.

BACKGROUND: Clinical trials that have assessed the best approach for treating under-nutrition in old age are scarce. OBJECTIVE: To determine the impact of an intensive nutritional intervention program led by a dietitian on the health and nutritional status of malnourished community dwelling older adults. METHODS: Sixty-eight eligible participants (age<75) were randomly assigned to a Dietetic Intervention Treatment (DIT), an intensive nutritional intervention led by a dietitian, or a Medical Treatment (MT), a physician-led standard care group, with an educational booklet regarding dietary requirements and recommendations for older adults. An additional 59 eligible participants who were unable to participate in the randomization were included as a non-randomized "untreated nutrition" group (UNG). RESULTS: Over the 6-month follow-up, the DIT group showed significant improvement in cognitive function (from 25.8±4.5 to 26.8±4, p=0.04), and depression score (from 7.3±3.9 to 5.4±3.9, p=0.04) compared with the change in the other 2 groups. The DIT group showed a significant improvement in intake of carbohydrates (+15% vs. +1% in the MT and +3% in the UNG), protein (+8% vs. +2% in the MT and -3% in the UNG), vitamin B6 (+20% vs. +7% in the MT and +8% in the UNG), and vitamin B1 (+22% vs. +11% in the MT and 0% in the UNG). The DIT group had a significantly lower cost of physician visits than the other 2 groups ($172.1±232.0 vs. $417.2±368.0 in the MT and $428.1±382.3 in the UNG, p=0.005). CONCLUSION: Intensive dietary intervention was moderately effective in lowering cost of services used and improving medical and nutritional status among community dwelling older adults.

Recruitment and activation of Raf-1 kinase by nitric oxide-activated Ras.

Nitric oxide (NO) and related species serve as cellular messengers in various physiological and pathological processes. The monomeric G protein, Ras, transduces multiple signaling pathways with varying biological responses. We have previously reported that NO triggers Ras activation and recruitment of an effector, phosphatidylinositol 3'-kinase (PI3K) and Ras-dependent activation of mitogen-activated protein (MAP) kinases which include extracellular signal regulated kinases (ERKs), c-Jun NH(2)-terminal kinase (JNK), and p38 MAP kinase. In this study, we further defined NO-activated Ras signaling pathways. We have identified Raf-1 as another effector recruited by NO-activated Ras in T lymphocytes. NO activation results in association of Ras and Raf-1 and is biologically significant, as we observe an NO-induced increase in Raf-1 kinase activity. Downstream to Raf-1 kinase lie MAP kinases and their subsequent downstream targets, transcription factors. We found that treatment of T lymphocytes with NO yielded phosphorylation of the transcription factor, Elk-1. This phoshorylation is dependent on NO binding to the cysteine 118 residue of Ras. By further delineating the pathway with pharmacological inhibitors, Elk-1 phosphorylation was also found to be dependent on PI3K and ERK. Moreover, NO triggered an increase in mRNA levels of the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha), which was ERK dependent. Thus, we have defined an NO-induced signaling pathway in T lymphocytes arising at the membrane where NO-activated Ras recruits Raf-1 and culminating in the nucleus where Elk-1 is phosphorylated and TNF-alpha messenger RNA is induced. This NO-activated Ras-mediated signaling pathway may play a critical role in Elk-1-induced transcriptional activation of T lymphocytes, host defense and inflammation.

Activation of c-Ha-Ras by nitric oxide modulates survival responsiveness in neuronal PC12 cells.

p21(c-Ha-Ras) (Ras) can be activated by the guanine nucleotide exchange factor mSOS1 or by S-nitrosylation of cysteine 118 via nitric oxide (NO). To determine whether these two Ras-activating mechanisms modulate distinct biological effects, a NO-nonresponsive Ras mutant (Ras(C118S)) was stably expressed in the PC12 cells, a cell line that generates NO upon nerve growth factor treatment. We report here that Ras(C118S) functions indistinguishably from wild type Ras in activating and maintaining the mSOS1- and Raf-1-dependent mitogen-activated protein kinase cascade necessary for neuronal differentiation. However, continuous (>5 days) exposure to nerve growth factor reveals that, in contrast to parental or wild-type Ras-overexpressing PC12 cells, Ras(C118S)-expressing PC12 cells cannot sustain the basal interaction between Ras and phosphatidylinositol 3-kinase. This results in spontaneous apoptosis of these cells despite the presence of nerve growth factor and serum. Thus unique downstream effector interactions and biological outcomes can be differentially modulated by distinct modes of Ras activation.

Reversible S-nitrosation and inhibition of HIV-1 protease.

Nitric oxide (*NO) is a short-lived free radical with many functions including vasoregulation, synaptic plasticity, and immune modulation and has recently been associated with AIDS pathology. Various pathophysiological conditions, such as viral infection, trigger inducible nitric oxide synthase (iNOS) to synthesize NO in the cell. NO-derived species can react with thiols of proteins and form nitrosothiol adducts. HIV-1 protease (HIV-PR) contains two cysteine residues, Cys67 and Cys95, which are believed to serve a regulatory function. We have found that HIV-PR is inactivated by nitric oxide produced in vitro by NO donors and by iNOS. Sodium nitroprusside inhibited HIV-PR by 70%, and S-nitroso-N-acetylpenicillamine completely inhibited the enzyme. Furthermore, iNOS generated sufficient NO to inhibit HIV-PR activity by almost 90%. This inactivation was reversed by the addition of reducing agents. Treatment of HIV-PR with NO donors and ritonavir (a competitive peptide inhibitor) indicates that NO exerts its effect through a site independent of the active site of HIV-PR. Using electrospray ionization mass spectrometry, we found that NO forms S-nitrosothiols on Cys67 and Cys95 of HIV-PR which directly correlate with a loss of activity. These data indicate that NO may suppress HIV-1 replication by directly inhibiting HIV-PR.

Targeting cysteine residues of human immunodeficiency virus type 1 protease by reactive free radical species.

Nitric oxide (NO) is a naturally occurring free radical with many functions. The oxidized form of NO, the nitrosonium ion, reacts with the thiol group of cysteine residues resulting in their modification to S-nitrosothiols. The human immunodeficiency virus type 1 (HIV-1) protease (HIV-PR) has two cysteine residues that are conserved amongst different viral isolates found in patients with acquired immunodeficiency syndrome (AIDS). In an active dimer, these residues are located near the surface of the protease. We have found that treatment of HIV-PR with different NO congeners results in loss of its proteolytic activity and simultaneous formation of S-nitrosothiols. Sodium nitroprusside inhibited HIV-PR up to 70% and S-nitroso-N-acetylpenicillamine completely inhibited the protease within 5 min of treatment. The pattern of inhibition by NO donors is comparable to its inhibition by N-acetyl pepstatin. Using electrospray ionization-mass spectrometry, we identified the modification of HIV-PR by NO as that of S-nitrosation. Our findings point towards a possible role of NO in mediating resistance to HIV-1 infection.

A redox-triggered ras-effector interaction. Recruitment of phosphatidylinositol 3'-kinase to Ras by redox stress.

Reactive free radical species are known to trigger biochemical events culminating in transcription factor activation and modulation of gene expression. The cytosolic signaling events triggered by free radicals that result in nuclear responses are largely unknown. Here we identify a signaling cascade triggered immediately upon redox activation of Ras. We examined two physiologically relevant models of redox signaling: 1) nitric oxide in human T cells, and 2) advanced glycation end product in rat pheochromocytoma cells. Reactive free radical species generated by nitric oxide donors and the interaction of advanced glycation end product with its receptor led to the recruitment of p85/p110 phosphatidylinositol 3'-kinase (PI3K) to the plasma membrane, where it associated directly with the effector domain of Ras and became activated. Only the p110beta and p110delta (but not p110alpha) catalytic subunits were recruited by redox-activated Ras. Activation of downstream targets of PI3K such as protein kinase B/Akt and mitogen-activated protein kinase was found to be PI3K dependent. Our study demonstrates that nitrosative and oxidative stressors trigger Ras-dependent and PI3K-regulated events in cells and define a biochemical pathway that is triggered by redox signaling.

Bradykinin B2-receptor activation augments norepinephrine exocytosis from cardiac sympathetic nerve endings. Mediation by autocrine/paracrine mechanisms.

We determined whether local bradykinin production modulates cardiac adrenergic activity. Depolarization of guinea pig heart sympathetic nerve endings (synaptosomes) with 1 to 100 mmol/L K+ caused the release of endogenous norepinephrine (10% to 50% above basal level). This release was exocytotic, because it depended on extracellular Ca2+, was inhibited by the N-type Ca(2+)-channel blocker omega-conotoxin and the protein kinase C inhibitor Ro31-8220, and was potentiated by the neuronal uptake-1 inhibitor desipramine. Typical of adrenergic terminals, norepinephrine exocytosis was enhanced by activation of prejunctional angiotensin AT1-receptors and attenuated by adrenergic alpha 2-receptors, adenosine A1-receptors, and histamine H3-receptors. Exogenous bradykinin enhanced norepinephrine exocytosis by 7% to 35% (EC50, 17 nmol/L), without inhibiting uptake 1. B2-receptor, but not B1-receptor, blockade antagonized this effect. The kininase II/angiotensin-converting enzyme inhibitor enalaprilat and the addition of kininogen or kallikrein enhanced norepinephrine exocytosis by approximately equal to 6% to 40% (EC50, 20 nmol/L) and approximately equal to 25% to 60%, respectively. This potentiation was prevented by serine protease inhibitors and was antagonized by B2-receptor blockade. Therefore, norepinephrine exocytosis is augmented when bradykinin synthesis is increased or when its breakdown is inhibited. This is the first report of a local kallikrein-kinin system in adrenergic nerve endings capable of generating enough bradykinin to activate B2-receptors in an autocrine/paracrine fashion and thus enhance norepinephrine exocytosis. This amplification process may operate in disease states, such as myocardial ischemia, associated with severalfold increases in local kinin concentrations.

G-protein-mediated signaling in cholesterol-enriched arterial smooth muscle cells. 1. Reduced membrane-associated G-protein content due to diminished isoprenylation of G-gamma subunits and p21ras.

Mechanisms contributing to altered heterotrimeric G-protein expression and subsequent signaling events during cholesterol accretion have been unexplored. The influence of cholesterol enrichment on G-protein expression was examined in cultured smooth muscle cells that resemble human atherosclerotic cells by exposure to cationized LDL (cLDL). cLDL, which increases cellular free and esterified cholesterol 2-fold and 10-fold, respectively, reduced the cell membrane content of Galphai-1, Galphai-2, Galphai-3, Gq/11, and Galphas. The following evidence supports the premise that the mechanism by which this occurs is due to reduced isoprenylation of the Ggamma-subunit. First, the inhibitory effect of cholesterol enrichment on the membrane content of Galphai subunits was found to be post-transcriptional, since the mRNA steady-state levels of Galphai(1-3) were unchanged following cholesterol enrichment. Second, the membrane expression of alpha and beta subunits was mimicked by cholesterol and 17-ketocholesterol, both of which inhibit HMG-CoA reductase. Third, inhibition of Galphai and Gbeta expression in cholesterol-enriched cells was overcome by mevalonate, the immediate product of HMG-CoA reductase. Fourth, pulse-chase experiments revealed that cholesterol enrichment did not reduce the degradation rate of membrane-associated Galphai subunits. Fifth, cholesterol enrichment also reduced membrane expression of Ggamma-5, Ggamma-7upper; these gamma subunits are responsible for trafficking of the heterotrimeric G-protein complex to the cell membrane as a result of HMG-CoA reductase-dependent post-translational lipid modification (geranylgeranylation) and subsequent membrane association. Cholesterol enrichment did not alter expression of G-gamma-5 mRNA, as assessed by reverse transcriptase polymerase chain reaction, supporting a post-transcriptional defect in Ggamma subunit expression. Fifth, cholesterol enrichment also reduced the membrane content of p21ras (a low molecular weight G-protein requiring farnesylation for membrane targeting) but did not alter the membrane content of the two proteins that do not require isoprenylation for membrane association&sbd;PDGF-receptor or p60-src. Reduced G-protein content in cholesterol-laden cells was reflected by reduced G-protein-mediated signaling events, including ATP-induced GTPase activity, thrombin-induced inhibition of cyclic AMP accumulation, and MAP kinase activity. Collectively, these results demonstrate that cholesterol enrichment reduces G-protein expression and signaling by inhibiting isoprenylation and subsequent membrane targeting. These results provide a molecular basis for altered G-protein-mediated cell signaling processes in cholesterol-enriched cells.

G-protein-mediated signaling in cholesterol-enriched arterial smooth muscle cells. 2. Role of protein kinase C-delta in the regulation of eicosanoid production.

PGI2 generation by the vessel wall is an agonist for cyclic-AMP-dependent cholesteryl ester hydrolysis. The process of enhanced PGI2 synthesis is stimulated, in part, by G-protein-coupled receptor ligands. Cellular cholesterol enrichment has been hypothesized to alter G-protein-mediated PGI2 synthesis. In the studies reported herein, cells generated PGI2 in response to AlF4-, GTPgammaS, and ATP in a dose-dependent manner. G-protein agonists stimulated eicosanoid production principally by activating phospholipase A2, but not phospholipase C. This is in contrast to PDGF, which stimulated phospholipase A2 and PLCgamma activities. Galphai subunits mediate G-protein agonist-induced PGI2 synthesis, since ATP- and PDGF-induced PGI2 synthesis was inhibited by pertussis toxin. Although cholesterol enrichment reduced arachidonic acid- and PDGF-induced PGI2 synthesis, cholesterol enrichment enhanced PGI2 release in response to AlF4-, GTPgammaS, and ATP. The enhancement of PGI2 release in cholesterol-enriched cells was augmented by mevalonate, which inhibits the ability of cholesterol enrichment to reduce membrane-associated G-protein subunits. Since cholesterol enrichment inhibited PDGF and AlF4--induced MAP kinase activity [Pomerantz, K., Lander, H. M., Summers, B., Robishaw, J. D., Balcueva, E. A., & Hajjar, D. P. (1997) Biochemistry 36, 9523-9531] (the major mechanism by which phospholipase A2 is activated), these results suggest that cholesterol enrichment induces other alternative signaling pathways leading to phospholipase A2 activation. A PKC-dependent pathway is described herein that is involved in enhanced eicosanoid production in cholesterol-enriched cells. This conclusion is supported by two observations: (1) G-protein-linked PGI2 production is inhibited by calphostin, and (2) cholesterol enrichment augments the specific translocation of the delta-isoform of PKC from the cytosol to the plasma membrane following treatment of cells with phorbol ester. These data support the concept that, in cells possessing normal levels of cholesterol, MAP-kinase-dependent pathways mediate eicosanoid synthesis in response to G-protein activation; however, under conditions of high cellular cholesterol levels, augmented G-protein-linked eicosanoid production results from enhanced PKCdelta activity.

Activation of the receptor for advanced glycation end products triggers a p21(ras)-dependent mitogen-activated protein kinase pathway regulated by oxidant stress.

Advanced glycation end products (AGEs) exert their cellular effects on cells by interacting with specific cellular receptors, the best characterized of which is the receptor for AGE (RAGE). The transductional processes by which RAGE ligation transmits signals to the nuclei of cells is unknown and was investigated. AGE-albumin, a prototypic ligand, activated p21(ras) in rat pulmonary artery smooth muscle cells that express RAGE, whereas nonglycated albumin was without effect. MAP kinase activity was enhanced at concentrations of AGE-albumin, which activated p21(ras) and NF-kappaB. Depletion of intracellular glutathione rendered cells more sensitive to AGE-mediated activation of this signaling pathway. In contrast, signaling was blocked by preventing p21(ras) from associating with the plasma membrane or mutating Cys118 on p21(ras) to Ser. Signaling was receptor-dependent, because it was prevented by blocking access to RAGE with either anti-RAGE IgG or by excess soluble RAGE. These data suggest that RAGE-mediated induction of cellular oxidant stress triggers a cascade of intracellular signals involving p21(ras) and MAP kinase, culminating in transcription factor activation. The molecular mechanism that triggers this pathway likely involves oxidant modification and activation of p21(ras).

A molecular redox switch on p21(ras). Structural basis for the nitric oxide-p21(ras) interaction.

We have identified the site of molecular interaction between nitric oxide (NO) and p21(ras) responsible for initiation of signal transduction. We found that p21(ras) was singly S-nitrosylated and localized this modification to a fragment of p21(ras) containing Cys118. A mutant form of p21(ras), in which Cys118 was changed to a serine residue and termed p21(ras)C118S, was not S-nitrosylated. NO-related species stimulated guanine nucleotide exchange on wild-type p21(ras), resulting in an active form, but not on p21(ras)C118S. Furthermore, in contrast to parental Jurkat T cells, NO-related species did not stimulate mitogen-activated protein kinase activity in cells transfected with p21(ras)C118S. These data indicate that Cys118 is a critical site of redox regulation of p21(ras), and S-nitrosylation of this residue triggers guanine nucleotide exchange and downstream signaling.

An essential role for free radicals and derived species in signal transduction.

It is well accepted that extracellular ligands trigger nuclear signals through a cascade of protein-protein interactions. Many of these pathways have been carefully defined and provide an important framework by which we can understand and intervene in the processes they initiate. Recent data in the literature indicate that many extracellular ligands generate and/or require reactive free radicals or derived species to successfully transmit their signals to the nucleus. Thus, a novel signaling mechanism akin to one solely dependent on protein-protein interactions may exist. Here, we review this information, identify both the sources and targets of free radicals generated by various growth factors and cytokines, discuss how specificity can be achieved, and explore the pathophysiological implications.

Differential activation of mitogen-activated protein kinases by nitric oxide-related species.

Many studies have identified nitric oxide (NO) and related chemical species (NOx) as having critical roles in neurotransmission, vasoregulation, and cellular signaling. Previous work in this laboratory has focused on elucidating the mechanism of NOx signaling in cells. We have demonstrated that NOx-induced activation of the guanine nucleotide-binding protein p21(ras) leads to nuclear translocation of the transcription factor NFkappaB. Here, we investigated whether intermediary signaling elements, namely the mitogen-activated protein (MAP) kinases, are involved in mediating NOx signaling. We found that NOx activates the extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK) subgroups of MAP kinases in human Jurkat T cells. JNK was found to be 100-fold more sensitive to NOx stimulation than p38 and ERK. In addition, the activation of JNK and p38 by NOx was more rapid than ERK activation. Depletion of intracellular glutathione augmented the NOx-induced increase in kinase activity. Furthermore, endogenous NO, generated from NO synthase, activated ERK, and NOx-induced MAP kinase activation was effectively blocked by the farnesyl transferase inhibitor alpha-hydroxyfarnesylphosphonic acid. These data support the hypothesis that critical signaling kinases, such as ERK, p38, and JNK, are activated by NO-related species and thus participate in NO signal transduction. These findings establish a role for multiple MAP kinase signaling pathways in the cellular response to NOx.

Activation of histamine H3-receptors inhibits carrier-mediated norepinephrine release during protracted myocardial ischemia. Comparison with adenosine A1-receptors and alpha2-adrenoceptors.

We previously showed that prejunctional histamine H3-receptors downregulate norepinephrine exocytosis, which is markedly enhanced in early myocardial ischemia. In the present study, we investigated whether H3-receptors modulate nonexocytotic norepinephrine release during protracted myocardial ischemia. In this setting, decreased pH(i) in sympathetic nerve endings sequentially leads to a compensatory activation of the Na+-H+ antiporter (NHE), accumulation of intracellular Na+, reversal of the neuronal uptake of norepinephrine, and thus carrier-mediated release of norepinephrine. Accordingly, norepinephrine overflow from isolated guinea pig hearts undergoing 20-minute global ischemia and 45-minute reperfusion was attenuated approximately 80% by desipramine (10 nmol/L) and 70% by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, 10 micromol/L), inhibitors of norepinephrine uptake and NHE, respectively. The H3-receptor agonist imetit (0.1 micromol/L) decreased carrier-mediated norepinephrine release by approximately 50%. This effect was blocked by the H3-receptor antagonist thioperamide (0.3 micromol/L), indicating that H-receptor activation inhibits carrier-mediated norepinephrine release. At lower concentrations, imetit (10 nmol/L) or EIPA (3 micromol/L) did not inhibit carrier-mediated norepinephrine release. However, a 25% inhibition occurred with imetit (10 nmol/L) and EIPA (3 micromol/L) combined. This synergism suggests an association between H-receptors and NHE. Conceivably, activation of H-receptors may lead to inhibition of NHE. In fact, alpha2-adrenoceptor activation, which is known to stimulate NHE, enhanced norepinephrine release, whereas alpha2-adrenoceptor blockade attenuated it. Furthermore, activation of adenosine A1-receptors markedly attenuated norepinephrine release, whereas their inhibition potentiated it. Because norepinephrine directly correlated with the severity of reperfusion arrhythmia and imetit reduced the incidence of ventricular fibrillation by 50%, our findings with H-receptor agonists may further the development of novel pharmacological means to reduce reperfusion arrhythmias in the clinical setting.

p21ras as a common signaling target of reactive free radicals and cellular redox stress.

Reactive free radicals have been implicated in mediating signal transduction by a variety of stimuli. We have investigated the role of p21ras in mediating free radical signaling. Our studies revealed that signaling by oxidative agents which modulate cellular redox status, such as H2O2, hemin, Hg2+, and nitric oxide was prevented in cells in which p21ras activity was blocked either through expression of a dominant negative mutant or by treating with a farnesyltransferase inhibitor, as assessed by NF-kappa B binding activity. Furthermore, the NF-kappa B response to these oxidative stress stimuli was found to be enhanced when cells from the human T cell line, Jurkat, were pretreated with L-buthionine-(S,R)-sulfoximine, an inhibitor of glutathione synthesis. We directly assayed p21ras and mitogen-activated protein kinase activities in Jurkat cells and found both of these signaling molecules to be activated in cells treated with the redox modulating agents. Blocking glutathione synthesis made cells 10- to 100-fold more sensitive to these agents. Finally, using recombinant p21ras in vitro, we found that redox modulators directly promoted guanine nucleotide exchange on p21ras. This study suggests that direct activation of p21ras may be a central mechanism by which a variety of redox stress stimuli transmit their signal to the nucleus.

v-Crk modulation of growth factor-induced PC12 cell differentiation involves the Src homology 2 domain of v-Crk and sustained activation of the Ras/mitogen-activated protein kinase pathway.

Nerve growth factor (NGF) and epidermal growth factor (EGF) elicit contrasting actions on PC12 pheochromocytoma cells; NGF causes neuronal differentiation, and EGF induces proliferation. However, ectopic expression of the Src homology 2 (SH2) and SH3-containing oncogenic adaptor protein v-Crk in PC12 cells results in EGF-inducible neuronal differentiation (Hempstead, B. L., Birge, R. B., Fajardo, J. E., Glassman, R., Mahadeo, D., Kraemer, R., and Hanafusa, H. (1994) Mol. Cell. Biol. 14, 1964-1971). Here we show that v-Crk complexes with both the tyrosine-phosphorylated EGF receptor and the Ras guanine nucleotide exchange factor SOS in PC12 cells and is involved in an pathway analogous to that of Grb2. Expression of v-Crk results in an enhanced and sustained activation of Ras and mitogen-activated protein (MAP) kinase following EGF or NGF stimulation, implying that v-Crk can couple divergent tyrosine kinase pathways to Ras. To investigate the causal relationship between EGF receptor binding, MAP kinase activation, and neurite outgrowth, we stably expressed two v-Crk SH2 point mutants, v-Crk(R273N) and v-Crk(H294R) in PC12 cells. Mutations within the SH2 domain of v-Crk block binding of v-Crk to the tyrosine phosphorylated EGF receptor, compromise v-Crk's ability to cause EGF-dependent neurite outgrowth, and act in a dominant negative manner for NGF-induced neurite outgrowth. However, the kinetics of MAP kinase activation in EGF- or NGF-treated v-Crk-(R273N)PC12 cells was comparable with that in v-CrkPC12 cells. These data are consistent with a model in which v-Crk regulates the strength of a tyrosine kinase signal leading to prolonged activation of Ras and MAP kinase. However, the experiments with the SH2 mutants suggest that sustained activation, by itself, may not be sufficient to switch the fate of v-CrkPC12 cells from proliferation toward differentiation.