David R. Kaplan

The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase

The serine/threonine protein kinase encoded by the Akt proto-oncogene is catalytically inactive in serum-starved primary and immortalized fibroblasts. Here we show that Akt and the Akt-related kinase AKT2 are activated by PDGF. The activation was rapid and specific, and it was abrogated by mutations in the Akt Pleckstrin homology (PH) domain. The Akt activation was also shown to depend on PDGFR beta tyrosines Y740 and Y751, which bind phosphatidylinositol 3-kinase (PI 3-kinase) upon phosphorylation. Moreover, Akt activation was blocked by the PI 3-kinase-specific inhibitor wortmannin and the dominant inhibitory N17Ras. Conversely, Akt activity was induced following the addition of phosphatidylinositol-3-phosphate to Akt immunoprecipitates from serum-starved cells in vitro. These results identify Akt as a novel target of PI 3-kinase and suggest that the Akt PH domain may be a mediator of PI 3-kinase signaling.

Neurotrophin signal transduction in the nervous system

Neurotrophins use two types of receptors, the Trk tyrosine kinase receptors and the p75 neurotrophin receptor (p75NTR), to regulate the growth, development, survival and repair of the nervous system. These receptors can either collaborate with or inhibit each others actions to mediate neurotrophin effects. The development and survival of neurons is thus based upon the functional interplay of the signals generated by Trk and p75NTR. In the past two years, the signaling pathways used by these receptors, including Akt and MAPK-induced signaling via Trk, and JNK, p53, and NF-kappaB signaling via p75NTR, have been identified. In addition, a number of novel p75NTR-interacting proteins have been identified that transmit growth, survival, and apoptotic signals.

Isolation of multipotent adult stem cells from the dermis of mammalian skin

We describe here the isolation of stem cells from juvenile and adult rodent skin. These cells derive from the dermis, and clones of individual cells can proliferate and differentiate in culture to produce neurons, glia, smooth muscle cells and adipocytes. Similar precursors that produce neuron-specific proteins upon differentiation can be isolated from adult human scalp. Because these cells (termed SKPs for skin-derived precursors) generate both neural and mesodermal progeny, we propose that they represent a novel multipotent adult stem cell and suggest that skin may provide an accessible, autologous source of stem cells for transplantation.

Neurotrophin-5: A novel neurotrophic factor that activates trk and trkB

In vertebrates, the formation and maintenance of neuronal connections are subject to regulation by multiple target-derived, diffusible (neurotrophic) factors. Here we describe the identification and characterization of a novel neurotrophic factor designated neurotrophin-5 (NT-5). NT-5 is structurally related to nerve growth factor and is expressed in embryonic as well as adult tissues. Recombinant NT-5 promotes the survival of peripheral sensory and sympathetic neurons and induces differentiation of the pheochromocytoma cell line PC12. NT-5 activates two trk-related tyrosine kinase receptors and shares these receptors with other neurotrophins. Activation of multiple receptors may permit a single neurotrophin to control target innervation by distinct neuronal populations. Receptor sharing could enable neurotrophic factors emanating from distinct targets to cooperate in regulating neurons with multiple connections.

A dermal niche for multipotent adult skin-derived precursor cells

A fundamental question in stem cell research is whether cultured multipotent adult stem cells represent endogenous multipotent precursor cells. Here we address this question, focusing on SKPs, a cultured adult stem cell from the dermis that generates both neural and mesodermal progeny. We show that SKPs derive from endogenous adult dermal precursors that exhibit properties similar to embryonic neural-crest stem cells. We demonstrate that these endogenous SKPs can first be isolated from skin during embryogenesis and that they persist into adulthood, with a niche in the papillae of hair and whisker follicles. Furthermore, lineage analysis indicates that both hair and whisker follicle dermal papillae contain neural-crest-derived cells, and that SKPs from the whisker pad are of neural-crest origin. We propose that SKPs represent an endogenous embryonic precursor cell that arises in peripheral tissues such as skin during development and maintains multipotency into adulthood.

Signal transduction by the neutrophin receptors

The neurotrophins signal cell survival, differentiation, growth cessation, and apoptosis through two cell surface receptors, the Trks and p75NTR (p75 neurotrophin receptor). Recent advances indicate that the particular events that are mediated by neurotrophins are dependent upon the cell type and the expression pattern of each neurotrophin receptor. For example, TrkA activation induces cell death of neural tumor cells, and survival and differentiation of neurons. Likewise, p75NTR, when activated in the absence of a strong Trk signal, induces apoptosis of neurons, while in the presence of Trk it enhances responses to neurotrophin. These differing responses point to a complex interplay between neurotrophin-stimulated survival, differentiation, and apoptosis pathways.

Trk receptors use redundant signal transduction pathways involving SHC and PLC-γ1 to mediate NGF responses

In response to NGF, the Trk receptor tyrosine kinase forms a complex with SHC, a protein that couples receptor tyrosine kinases to p21ras. Complex formation between Trk and SHC, SHC tyrosine phosphorylation, and association of SHC with Grb2 were mediated by autophosphorylation at Y490 in Trk [sequence: see text]. To determine the role of SHC and other Trk substrates in NGF signaling, Trk receptors with mutations in Y490 and Y785 (the PLC-gamma 1 association site) were introduced into PC12nnr5 cells. NGF treatment of PC12nnr5 cells expressing Trk with mutations in either substrate-binding site resulted in normal neurite outgrowth and Erk1 activity and tyrosine phosphorylation. However, PC12nnr5 cells expressing Trk with mutations at both sites failed to stably extend neurites and efficiently induce Erk1 activity and tyrosine phosphorylation in response to NGF. We postulate that Trk receptors can activate Erk1 by either SHC- or PLC-gamma 1-dependent signaling pathways. These results suggest a model whereby Trk receptors utilize at least partially redundant signal transduction pathways to mediate NGF responses.

Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity

The phosphorylation of proteins on tyrosine in vivo and in vitro was examined in 3T3 cells stimulated by platelet-derived growth factor (PDGF) and transformed by polyoma middle T antigen (MTAg) by using an antibody directed against phosphotyrosine (P-tyr). Two common events were observed upon PDGF stimulation or MTAg transformation of cells: the appearance in the immunoprecipitates of an 85 kd phosphoprotein, and increased phosphatidylinositol (PI) kinase activity. In PDGF-stimulated cells, the 85 kd phosphoprotein and PI kinase activity appeared rapidly, within 1 min of growth factor addition. The PI kinase activity and 85 kd phosphorylation were also increased in anti-P-tyr immunoprecipitates from cells transformed by v-fms and v-sis, but not by SV40 T antigen. The presence of the tyrosine-phosphorylated 85 kd protein correlated with PI kinase activity during several purification steps. These results suggest that the 85 kd phosphoprotein, a putative PI kinase, is a substrate for both the PDGF receptor and MTAg/pp60c-src tyrosine kinase activities.

The TrkB-Shc Site Signals Neuronal Survival and Local Axon Growth via MEK and PI3-Kinase

To determine how signals emanating from Trk transmit neurotrophin actions in primary neurons, we tested the ability of TrkB mutated at defined effector binding sites to promote sympathetic neuron survival or local axon growth. TrkB stimulated signaling proteins and induced survival and growth in a manner similar to TrkA. TrkB mutated at the Shc binding site supported survival and growth poorly relative to wild-type TrkB, whereas TrkB mutated at the PLC-gamma1 binding site supported growth and survival well. TrkB-mediated neuronal survival was dependent on P13-kinase and to a lesser extent MEK activity, while growth depended upon both MEK and P13-kinase activities. These results indicate that the TrkB-Shc site mediates both neuronal survival and axonal outgrowth by activating the P13-kinase and MEK signaling pathways.

p75LNGFR regulates Trk signal transduction and NGF-induced neuronal differentiation in MAH cells

We have examined NGF-induced signal transduction events and neuronal differentiation in MAH cells, a neuronal progenitor cell line, in which the expression of the two NGF receptors, p140trk (Trk) and p75LNGFR (p75), has been independently manipulated. Coexpression of a large molar excess of p75 substantially enhances the NGF-induced tyrosine autophosphorylation of Trk, compared with cells expressing Trk alone. MAH cells expressing both Trk and p75 stop dividing and acquire a mature neuronal morphology more rapidly and with greater efficiency than MAH cells expressing Trk alone. These biochemical and biological influences of p75 are not observed using a mutant form of NGF that binds Trk but not p75. These data provide evidence that p75 can modulate signal transduction through Trk in a neuronal progenitor cell context and that such modulation has functional consequences for the neuronal differentiation pathway induced by NGF.