Stephen P. Soltoff

Oncogenes and signal transduction

The purpose of this review is to incorporate recent discoveries into a general biochemical pathway by wich the protein products of the oncogenes send signals from the cell surface to the nucleus .The protein-tyrosine kinase oncogenes will be the primary focus of the review .However, biochemical connections between the protein tyrosine kinases and oncoproteins of the Ras,Raf,Fos,Jun,and Rel families as well as the protein kinase C family are also discussed .

ErbB3 is involved in activation of phosphatidylinositol 3-kinase by epidermal growth factor.

Conflicting results concerning the ability of the epidermal growth factor (EGF) receptor to associate with and/or activate phosphatidylinositol (PtdIns) 3-kinase have been published. Despite the ability of EGF to stimulate the production of PtdIns 3-kinase products and to cause the appearance of PtdIns 3-kinase activity in antiphosphotyrosine immunoprecipitates in several cell lines, we did not detect EGF-stimulated PtdIns 3-kinase activity in anti-EGF receptor immunoprecipitates. This result is consistent with the lack of a phosphorylated Tyr-X-X-Met motif, the p85 Src homology 2 (SH2) domain recognition sequence, in this receptor sequence. The EGF receptor homolog, ErbB2 protein, also lacks this motif. However, the ErbB3 protein has seven repeats of the Tyr-X-X-Met motif in the carboxy-terminal unique domain. Here we show that in A431 cells, which express both the EGF receptor and ErbB3, PtdIns 3-kinase coprecipitates with the ErbB3 protein (p180erbB3) in response to EGF. p180erbB3 is also shown to be tyrosine phosphorylated in response to EGF. In contrast, a different mechanism for the activation of PtdIns 3-kinase in response to EGF occurs in certain cells (PC12 and A549 cells). Thus, we show for the first time that ErbB3 can mediate EGF responses in cells expressing both ErbB3 and the EGF receptor.

The C2 Domain of PKCδ Is a Phosphotyrosine Binding Domain

Summary In eukaryotic cells, the SH2 and PTB domains mediate protein-protein interactions by recognizing phosphotyrosine residues on target proteins. Here we make the unexpected finding that the C2 domain of PKCδ directly binds to phosphotyrosine peptides in a sequence-specific manner. We provide evidence that this domain mediates PKCδ interaction with a Src binding glycoprotein, CDCP1. The crystal structure of the PKCδ C2 domain in complex with an optimal phosphopeptide reveals a new mode of phosphotyrosine binding in which the phosphotyrosine moiety forms a ring-stacking interaction with a histidine residue of the C2 domain. This is also the first example of a protein Ser/Thr kinase containing a domain that binds phosphotyrosine.

p120cbl is a major substrate of tyrosine phosphorylation upon B cell antigen receptor stimulation and interacts in vivo with Fyn and Syk tyrosine kinases, Grb2 and Shc adaptors, and the p85 subunit of phosphatidylinositol 3-kinase.

We and others have demonstrated that the c-cbl proto-oncogene product is one of the earliest targets of tyrosine phosphorylation upon T cell receptor stimulation. Given the similarities in the B and T lymphocyte antigen receptors, and the induction of pre-B leukemias in mice by the v-cbl oncogene, we examined the potential involvement of Cbl in B cell receptor signaling. We demonstrate prominent and early tyrosine phosphorylation of Cbl upon stimulation of human B cell lines through surface IgM. Cbl was associated in vivo with Fyn and, to a lesser extent, other Src family kinases. B cell activation also induced a prominent association of Cbl with Syk tyrosine kinase. A substantial fraction of Cbl was constitutively associated with Grb2 and this interaction was mediated by Grb2 SH3 domains. Tyrosine-phosphorylated Shc, which prominently associated with Grb2, was detected in association with Cbl in activated B cells. Thus, Grb2 and Shc adaptors, which associate with immunoreceptor tyrosine based activation motifs, may link Cbl to the B cell receptor. B cell activation also induced a prominent association between Cbl and the p85 subunit of phosphatidylinositol (PI) 3-kinase resulting in the association of a substantial fraction of PI 3-kinase activity with Cbl. Thus, Cbl is likely to play an important role to couple the B cell receptor to the PI 3-kinase pathway. Our results strongly suggest a role for p120 in signaling downstream of the B cell receptor and support the idea that Cbl participates in a general signal transduction function downstream of the immune cell surface receptors.

Related Adhesion Focal Tyrosine Kinase and the Epidermal Growth Factor Receptor Mediate the Stimulation of Mitogen-activated Protein Kinase by the G-protein-coupled P2Y2 Receptor PHORBOL ESTER OR [Ca2+] i ELEVATION CAN SUBSTITUTE FOR RECEPTOR ACTIVATION

The activation of growth factor receptors and receptors coupled to heterotrimeric guanine nucleotide-binding proteins (G-proteins) can increase mitogen-activated protein (MAP) kinase activity in many cells. Previously, we demonstrated that the activation of G-protein-coupled P2Y2 receptors by extracellular ATP and UTP stimulated MAP (p42 ERK2) kinase by a mechanism that was dependent on the elevation of [Ca2+] i and the activation of related adhesion focal tyrosine kinase (RAFTK) (also called PYK2, CAKβ, and CADTK) and protein kinase C (PKC). Here, we examine further the signaling cascade between the P2Y2 receptor and MAP kinase. MAP kinase was transiently activated by exposure of PC12 cells to UTP. UTP, ionomycin, and phorbol ester (phorbol 12-myristate 13-acetate) increased MAP kinase activity and also promoted the tyrosine phosphorylation of RAFTK, the epidermal growth factor (EGF) receptor, SHC, and p120 cbl . Down-regulation of PKC and inhibition of the elevation of [Ca2+] i , conditions that block the activation of MAP kinase, also blocked the increases in the tyrosine phosphorylation of RAFTK and the EGF receptor. AG1478, a tyrphostin selective for the EGF receptor, reduced the activation of MAP kinase, the tyrosine phosphorylation of SHC, the association of Grb2 with SHC, and the tyrosine phosphorylation of the EGF receptor and p120 cbl but did not block the tyrosine phosphorylation of RAFTK. The similar effects of UTP, ionomycin, and phorbol 12-myristate 13-acetate (PMA) on these signaling proteins demonstrate that the two signaling molecules from phosphatidylinositol 4,5-bisphosphate hydrolysis ([Ca2+] i , from inositol 1,4,5-trisphosphate production, and diacylglycerol) can individually initiate the activation of MAP kinase in an EGF receptor-dependent manner. These results demonstrate that the P2Y2 receptor-mediated transactivation of the EGF receptor occurs at a point downstream of RAFTK and indicate that the EGF receptor is required for P2Y2 receptor-mediated MAP kinase activation. Although P2Y2 and EGF receptors may both activate a similar multiprotein signaling cascade immediately upstream of MAP kinase, the P2Y2 receptor appears to uniquely utilize [Ca2+] i , PKC, and, subsequently, RAFTK.

Extracellular Calcium and Platelet-derived Growth Factor Promote Receptor-mediated Chemotaxis in Osteoblasts through Different Signaling Pathways

The discovery of a calcium receptor has stimulated interest in the signaling events underlying extracellular calcium ([Ca2+]o)-induced cell-specific responses. In osteoblasts, elevated levels of extracellular calcium mediate both mitogenesis and chemotaxis. Here we provide evidence that [Ca2+]o-stimulated chemotaxis of MC3T3-E1 osteoblast-like cells involves a G-protein-linked calcium-sensing receptor. [Ca2+]o promotes chemotaxis in a concentration-dependent manner. Pertussis toxin blocked almost all of [Ca2+]o-stimulated chemotaxis but had only a small effect on platelet-derived growth factor (PDGF)-stimulated chemotaxis. Consistent with the signaling model for PDGF-mediated chemotaxis, activation of phospholipase C played a critical role in [Ca2+]o-initiated chemotaxis: U-73122, an inhibitor of the activation of phospholipase C, blocked approximately 50% of PDGF-stimulated chemotaxis but blocked nearly all of the [Ca2+]o-stimulated chemotaxis. Down-regulation of protein kinase C also blocked about 50% of PDGF-stimulated chemotaxis but did not block [Ca2+]o-stimulated chemotaxis. Thus, unlike PDGF-mediated chemotaxis, chemotaxis stimulated by [Ca2+]o does not appear to require protein kinase C activation. This finding suggests events downstream of inositol 1,4,5-trisphosphate production rather than diacylglycerol production are critical to [Ca2+]o-promoted chemotaxis of MC3T3-E1 cells. The signal transduction mechanism underlying PDGF-induced chemotaxis involves the activation of phosphoinositide 3-kinase, as judged by the in vivo production of phosphatidylinositol 3,4-diphosphate and 3,4,5-trisphosphate and the partial sensitivity of chemotaxis to wortmannin, an inhibitor of phosphoinositide 3-kinase. In contrast, [Ca2+]o-stimulated chemotaxis was not blocked by wortmannin and elevations in [Ca2+]o did not increase the production of lipid products of phosphoinositide 3-kinase. Overall, [Ca2+]o-promoted chemotaxis of osteoblasts appears to utilize a unique signaling mechanism via a calcium-sensing receptor.

Phosphatidylinositol 3-kinase-dependent activation of trypsinogen modulates the severity of acute pancreatitis

Intra-acinar cell activation of digestive enzyme zymogens including trypsinogen is generally believed to be an early and critical event in acute pancreatitis. We have found that the phosphatidylinositol 3-kinase inhibitor wortmannin can reduce the intrapancreatic activation of trypsinogen that occurs during two dissimilar experimental models of rodent acute pancreatitis, secretagogue- and duct injection-induced pancreatitis. The severity of both models was also reduced by wortmannin administration. In contrast, the NF-kappa B activation that occurs during the early stages of secretagogue-induced pancreatitis is not altered by administration of wortmannin. Ex vivo, caerulein-induced trypsinogen activation is inhibited by wortmannin and LY294002. However, the cytoskeletal changes induced by caerulein were not affected by wortmannin. Concentrations of caerulein that induced ex vivo trypsinogen activation do not significantly increase phosphatidylinositol-3,4-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate levels or induce phosphorylation of Akt/PKB, suggesting that class I phosphatidylinositol 3-kinases are not involved. The concentration of wortmannin that inhibits trypsinogen activation causes a 75% decrease in phosphatidylinositol 3-phosphate, which is implicated in vesicle trafficking and fusion. We conclude that a wortmannin-inhibitable phosphatidylinositol 3-kinase is necessary for intrapancreatic activation of trypsinogen and regulating the severity of acute pancreatitis. Our observations suggest that phosphatidylinositol 3-kinase inhibition might be of benefit in preventing acute pancreatitis.

Intracellular respiratory dysfunction and cell injury in short-term anoxia of rabbit renal proximal tubules.

The effects of short-term anoxia and hypoxia were studied in a rabbit proximal renal tubule suspension in order to avoid the hemodynamic consequences of clamp-induced ischemia. The suspension was subjected to anoxia for 10-40 min and the effects on a number of cellular transport and respiratory parameters were monitored. Cellular respiration was measured upon addition of nystatin (Nys) to maximally stimulate Na pump activity. Mitochondrial respiration was measured in the tubules by addition of digitonin and ADP to obtain the state 3 respiratory rate. The release of lactate dehydrogenase (LDH) was measured as an index of plasma membrane damage. The cellular contents of K and Ca were also measured. Results show that 10 and 20 min of anoxia partially inhibited Nys-stimulated and mitochondrial respiration, and partially decreased the K contents, but all these effects were largely reversible after 20 min of reoxygenation. After 40 min of anoxia and 20 min of reoxygenation, all these variables remained irreversibly inhibited: Nys-stimulated respiration by 54%, mitochondrial respiration by 50%, K content by 42%, and LDH release was 40% of total. Ca content decreased slightly during anoxia, but increased up to fourfold during severe hypoxia; the excess Ca was released during the first 10 min of reoxygenation. The degree of respiratory impairment was identical during anoxia or hypoxia, suggesting that Ca accumulation was not associated with the impairment. Decreasing the extracellular Ca to 2.5 microM decreased LDH release significantly during anoxia, suggesting that plasma membrane damage during anoxia may be associated with increased intracellular free Ca. Addition of Mg-adenosine triphosphate during anoxia dramatically improved recovery of all the measured parameters after the anoxic period.

Mechanisms whereby exogenous adenine nucleotides improve rabbit renal proximal function during and after anoxia.

When a suspension of rabbit proximal tubules is subjected to anoxia, ATP falls by 80-90% during 40 min of anoxia, and upon reoxygenation (reox) the cells only recover 25-50% of their initial ATP. Addition of Mg-ATP (magnesium chloride-treated ATP), Mg-ADP, or Mg-AMP (five aliquots of 200 nmol/ml added 10 min apart) during anoxia causes complete recovery of ATP levels, and respiratory and transport function after 40 min of reox. Similar additions of adenosine (ADO), or inosine (INO), or Mg-ATP only during reox are less effective. Lactate dehydrogenase (LDH) release after 40 min of anoxia is 30-40% under control conditions, only 10-15% when adenine nucleotides or ADO are added during anoxia, and 20% when INO is added, suggesting that these additions may stabilize the plasma membrane during anoxia and help preserve cellular integrity. During reox, recovery may depend on the entry of ATP precursors and, therefore, we explored the mechanism whereby exogenous ATP increases the intracellular ATP content. Additions of Mg-ATP, Mg-ADP, or Mg-AMP to continuously oxygenated tubules increase cellular ATP content three- to fourfold in 1 h. The added ATP and ADP are rapidly degraded to AMP, and more slowly to ADO, INO, and hypoxanthine. Furthermore, the ATP-induced increase in cellular ATP is abolished by the exogenous addition of adenosine deaminase, which converts extracellular ADO to INO. These results suggest that the increase in cellular ATP requires extracellular ADO. The ADO obtained from the breakdown of AMP may be preferentially transported into the renal cells to be resynthesized into cellular AMP and ATP.

Two distinct cytosolic calcium responses to extracellular ATP in rat parotid acinar cells.

1 Increasing concentrations of ATP (0.5 μm‐300 μm) produced a biphasic increase in intracellular calcium concentration [Ca]i in rat parotid acinar cells, reflecting two distinct Cai responses to extracellular ATP. 2 In the absence of Mg2+ (with 3 mm CaCl2 in the buffer solution), the more sensitive response was maximal at 3–5 μm and was not further increased by 30 μm ATP. This response to ATP was not well maintained and was blocked by ADP (0.5 mm). A second, much larger increase in Cai was observed on addition of 300 μm ATP. This larger effect, which we have described previously, appears to be mediated by ATP4–, and was selectively reversed by 4,4′‐di‐isothiocyanato‐dihydrostilbene‐2,2′‐disulphonate as well as by high concentrations of α,β‐methylene ATP. 3 Among ATP analogues, only the putative P2Z agonist, 3′‐0‐(4‐benzoyl)benzoyl‐ATP distinguished between the two responses. This analogue was at least 10 fold more potent than ATP in stimulating the ATP4–‐response, but did not evoke the more sensitive response. The agonist potency series for both responses to ATP was identical for other analogues examined (ATP>ATP‐γS = 2‐methylthio ATP (a P2y‐selective agonist) 72>ADP, ITP and α,β‐methylene ATP (a P2X‐selective agonist)). 4 Although the effect of ATP4– could best be characterized as a P2Z‐type purinoceptor response, this effect was strongly and selectively blocked by reactive blue 2, a putative P2y‐purinoceptor antagonist. Reactive blue 2 may bind to and block P2Z purinoceptors since [γ32P]‐ATP binding to parotid cells was inhibited by this compound. 5 In contrast to the response to ATP4–, the more sensitive response to ATP was potentiated by reactive blue 2 and was less affected by increases in external Mg2+ and Ca2+. 6 Parasympathetic denervation selectively increased the more sensitive response, suggesting that it may be physiologically regulated.