Transient association of the phosphotyrosine phosphatase SHP-2 with TrkA is induced by nerve growth factor.

Nerve growth factor (NGF) treatment of rat PC12 pheochromocytoma cells results in an increase in the tyrosine phosphorylation of the NGF receptor, TrkA, leading to differentiation to a neuronal phenotype. Dephosphorylation by protein tyrosine phosphatases (PTPases) is thought to play an important role in regulating this signaling pathway. To identify PTPases that are recruited to the activated TrkA receptor, we used an in-gel PTPase assay to examine the presence of PTPases in TrkA immunoprecipitates. The Src homology 2 domain containing PTPase SHP-2 was found to associate transiently with TrkA following receptor activation, reaching a peak after 1 min of NGF treatment and then decreasing rapidly. The association of SHP-2 with TrkA was accompanied by the tyrosine phosphorylation of SHP-2 and an association of SHP-2 with multiple tyrosine-phosphorylated proteins. In addition, the PTPase activity in SHP-2 immunoprecipitates increased greater than twofold after 1 min of NGF treatment. This is the first demonstration that the association of SHP-2 with TrkA is induced by NGF and that this association leads to SHP-2 activation and tyrosine phosphorylation. We conclude that SHP-2 plays a significant role in early biochemical events in TrkA-mediated signal transduction.

SHP-2 is involved in neurotrophin signaling.

To investigate the involvement of SHP-2 in the signal transduction pathway stimulated by neurotrophins, the association of SHP-2 with components of the pathway was examined. Following NGF stimulation of PC12 cells, SHP-2 was found to be associated with the p85 subunit of PI3-kinase and the Shc proteins. In retinoic acid-differentiated SH-SY5Y cells and primary cultures of rat cortical neurons, BDNF treatment similarly caused the association of SHP-2 with p85. In addition, a tyrosine-phosphorylated protein, which is probably TrkB, was coimmunoprecipitated with SHP-2 in both cultures. These results show that SHP-2 becomes associated with signaling proteins after treatment with neurotrophins and suggest that SHP-2 plays a fundamental role in neurotrophin signaling.

In Aplysia sensory neurons, the neuropeptide SCPB and serotonin differ in efficacy both in modulating cellular properties and in activating adenylyl cyclase: implications for mechanisms underlying presynaptic facilitation.

The facilitatory transmitters serotonin (5-HT) and the molluscan neuropeptides SCPA and SCPB both activate adenylyl cyclase in Aplysia mechanosensory neurons and produce multiple modulatory effects that contribute to increasing transmitter release from these cells. This enhancement of transmitter release from sensory neurons contributes to increased behavioral response during sensitization and classical conditioning in Aplysia. Recently, specific examples of modulation in these sensory neurons have been described that are more effectively initiated by 5-HT than by the SCPs. For example, in the present study, 5-HT produces 55% greater broadening of the normal sensory neuron action potential than did SCPB. These differences in the modulatory actions of the facilitatory transmitters have been interpreted as suggesting that 5-HT produces its modulatory effects at least partly via a cAMP-independent mechanism. However, we have found that the two types of facilitatory transmitters are not equally effective in activating adenylyl cyclase. In both whole CNS membranes and sensory neuron membranes, SCPB was less effective than 5-HT in stimulating adenylyl cyclase activity measured in steady state assays. Because electrophysiological experiments suggested that the response to the SCPs desensitizes rapidly, we further compared cyclase stimulation in perfused membrane assays that enable continuous monitoring of cyclase activity; however we observed that 5-HT was also more effective than SCPB in stimulating cyclase at the onset of transmitter exposure. We discuss the possibility that lower peak stimulation of cyclase by SCPB and a faster rate of desensitization could account for some of the differences between the SCPs and 5-HT in modulating sensory neurons.

cAMP modulates multiple K+ currents, increasing spike duration and excitability in Aplysia sensory neurons.

Enhancement of the defensive withdrawal reflex of Aplysia involves a prolongation of the action potentials of mechanosensory neurons, which contributes to facilitation of transmitter release from these cells. Recent reports have suggested that whereas cAMP-dependent modulation of K+ current increases sensory neuron excitability, a cAMP-independent decrease in K+ current may increase the action potential duration and, thus, facilitate transmitter release. We have tested this proposal using Walsh cAMP-dependent protein kinase inhibitor or activators of the cAMP cascade and found that cAMP plays a major role in the spike-broadening effects of facilitatory transmitter; however, broadening requires higher levels of activation of the cAMP-dependent kinase than does increasing excitability. A steeply voltage-dependent transient K+ current, termed IKV,early, and the slowly activating S-type K+ (S-K+) current are both reduced by activation of the cAMP cascade, although with different sensitivities to the second messenger, enabling excitability and spike duration to be regulated independently. Differences in cAMP sensitivity also suggested that the originally described S-K+ current actually consists of two independent components, a slowly activating component and a time-independent, "steady-state" current that is activated at rest.

Reversal of synaptic depression by serotonin at Aplysia sensory neuron synapses involves activation of adenylyl cyclase.

Facilitation of the monosynaptic connection between siphon sensory neurons and gill and siphon motor neuron contributes to sensitization and dishabituation of the gill and siphon withdrawal reflex in Aplysia. The facilitatory transmitter serotonin (5-HT) initiates two mechanisms that act in parallel to increase transmitter release from siphon sensory neurons. 5-HT acts, at least partly through cAMP, to broaden the presynaptic action potential. 5-HT also initiates a second process that facilitates depressed sensory neuron synapses by a mechanism independent of changes in action potential duration. Recent experiments indicated that either of two protein kinases, cAMP-dependent protein kinase A and protein kinase C, are capable of effectively activating this second facilitatory mechanism, restoring synaptic transmission in depressed synapses. We have used the adenylyl cyclase inhibitor SQ 22,536 [9-(tetrahydro-2-furyl)adenine or THFA] to explore the contribution of cAMP to the reversal of synaptic depression. THFA effectively inhibited both adenylyl cyclase activity in vitro and known cyclase-mediated effects in intact sensory neurons. THFA also completely blocked facilitation of depressed synapses by 5-HT. These results suggest that adenylyl cyclase plays a critical role in the reversal of synaptic depression that contributes to dishabituation in this system.

Surgical treatment of extramammary Paget's disease. A report of six cases and a reexamination of Mohs micrographic surgery compared with conventional surgical excision.

Extramammary Paget's disease (EMPD) is a rare cutaneous malignancy, usually on the genitalia, that almost always extends beyond clinically apparent margins. Recurrences after standard methods of surgical excision are notoriously frequent; effective treatment with Mohs micrographic surgery was first reported in 1979. It has since been suggested this malignancy may be multifocal, and reports of recurrences after resection with micrographic surgery have appeared. The authors report six cases treated with Mohs surgery, two of which recurred. They also present data on 42 additional cases obtained from a written survey of members of the American College of Mohs Micrographic Surgery and comparison cases selected from the literature. The recurrence rate after micrographic surgery appears to be at least as low as that after conventional surgical excision with vertical frozen section or paraffin section margin control. Mohs micrographic surgery allows for maximal tissue sparing of critical anatomic structures and is performed under local anesthesia as an outpatient; because of this, it may be superior to conventional surgical excision. A scheme for management of this malignancy is presented. Surgeons should be aware radical excision is not needed for most cases of extramammary Paget's disease and very long-term patient follow-up is required.

Hemidesmosomes, collagen VII, and intermediate filaments in basal cell carcinoma.

We have undertaken an analysis of hemidesmosomes (HD) and their associated structures, intermediate filaments (IF) and anchoring fibrils (AF), in various types of basal cell carcinoma (BCC). Using a combination of electron microscopy and immunofluorescence microscopy we show that there is a correlation between the loss of HD and tumor type (i.e., in solid and infiltrative BCC hemidesmosomes are present, sometimes in reduced numbers), while there appears to be a lack of hemidesmosomes in cells of sclerosing specimens. Moreover, even though there is a loss of cytoplasmic constituents of the HD in sclerosing forms of BCC, this is not the case with regard to collagen VII, a component of AF, which are normally associated with the extracellular side of the HD. Collagen VII is localized to the basement membrane zone of tumor cells in the absence of the cytoplasmic constituents of HD. Furthermore, deposits of collagen VII occur in the connective tissue close to tumor cell populations in all but one of the BCC specimens we analyzed. In addition to modifications in HD and AF in BCC tissue, there are changes in the cytoskeletal elements of both tumor cells and the normal appearing epidermis that overlies tumor areas. In sclerosing BCC microfilaments are commonly observed along the basal portions of tumor cells where they abut the connective tissue. IF are often found interacting with these microfilaments. Indirect immunofluorescence analysis of tumor tissue using a monoclonal keratin antibody preparation, AE1, which in normal epidermis stains basal cells, reveals that AE1 antibodies only weakly stain tumor cells. Moreover, in the epidermis that overlies tumor cell regions AE1 antibodies stain suprabasal cells and not basal cells. This change in staining pattern generated by AE1 antibodies appears to depend upon the proximity of tumor cells. These results are discussed in relation to the organization of the HD and its associated AF and IF. The possibility that HD, IF, and AF antibody preparations may be of diagnostic use is raised.