In this work, we examined the role of the SHP-1 pTyr phosphatase in neurotrophin-mediated cell survival and signal transduction. Our results indicate that SHP-1 is a TrkA phosphatase in PC12 cells and in sympathetic neurons in culture and in vivo, and that it functions to ensure low levels of basal TrkA activation and to attenuate long-term TrkA signaling in the presence of NGF. These conclusions are supported by four findings. First, we show that SHP-1 dephosphorylated TrkA in vivo and in vitro, and that dephosphorylation was predominantly at sites that controlled TrkA activity (Y674 and Y675). Second, overexpression of SHP-1 in sympathetic neurons and PC12 cells resulted in apoptosis as a consequence of TrkA dephosphorylation. Third, inhibition of endogenous SHP-1 activity was sufficient to support NGF-independent neuronal survival as a consequence of enhanced basal TrkA phosphorylation and downstream Akt activation. Inhibition of endogenous SHP-1 also led to sustained and elevated TrkA activation in the presence of NGF. Fourth, sympathetic neuron number was higher in mice genetically deficient in SHP-1, presumably as a consequence of enhanced TrkA activation during the period of naturally occurring neuronal death; neurons from these mice survived in limiting amounts of NGF. Together, these results argue that SHP-1 is a key negative regulator of TrkA-initiated signal transduction, and that it mediates this negative regulation largely at the level of the TrkA receptor. Such regulation is critical for maintaining the trophic factor dependence of at least one population of neurons during the naturally occurring cell death period, a dependence that is essential for establishing appropriate neuronal connectivity.
How does SHP-1 regulate TrkA activity? We propose that SHP-1 regulates both basal and NGF-stimulated TrkA activity. Because inhibition of endogenous SHP-1 stimulates the NGF-independent phosphorylation of TrkA, SHP-1 can regulate the basal, nonliganded activity of TrkA. TrkA activity, in the absence of NGF, is thus normally controlled and suppressed by SHP-1 activity. In the presence of NGF, TrkA is efficiently dimerized and hyperactivated, and TrkA tyrosine kinase activity predominates over basal SHP-1 tyrosine phosphatase activity. The enhanced TrkA activity results in receptor transphosphorylation, followed by recruitment of cytoplasmic signaling proteins to TrkA transphosphorylation sites, and TrkA-induced tyrosine phosphorylation of these substrates that in turn stimulates survival and growth pathways. However, SHP-1 is also recruited to and stimulated by NGF-bound TrkA, resulting in an increase in SHP-1 tyrosine phosphatase activity. The increase in SHP-1 tyrosine phosphatase activity would result in an attenuation of TrkA activity. Thus, we propose a model whereby SHP-1 either directly or indirectly associates with TrkA, resulting in an increase in SHP-1 activity followed by dephosphorylation of TrkA at the Y674 and Y675 sites; a similar mechanism is used by the tyrosine phosphatase PTP1B to regulate the insulin receptor (Salmeen et al., 2000
). The dephosphorylation of these sites results in decreased TrkA biochemical and biological activity (Cunningham et al., 1997
) and subsequent decreased activation of NGF-signaling proteins. Therefore, we suggest that SHP-1 has two functions: (1) to keep TrkA in an “off” state in the absence of ligand, and (2) to modulate TrkA activity after dimerization and activation of TrkA by NGF.
What is the role of SHP-1 during sympathetic neuron development? We propose that SHP-1 has two functions: (1) to control TrkA activity in the absence of NGF, and (2) to “fine-tune” TrkA-mediated survival signals in the presence of NGF. Correct neuron number during sympathetic development is dependent on the functional interplay of TrkA-induced survival signals and p75NTR
-induced apoptotic signals (Kaplan and Miller, 2000
; Majdan et al., 2001
). Mice deficient in TrkA lack most sympathetic neurons, whereas mice deficient in p75NTR
have twice the number of sympathetic neurons per ganglia in the SCG at P20. me/me
mice that lack SHP-1 have 35% more neurons than wt mice ( A), indicating that SHP-1 functions during development to either suppress TrkA activity or the activity of other apoptotic signals. On the basis of our work in cultured SCG neurons, we favor the former hypothesis. In particular, we propose that SHP-1 is essential to keep TrkA off in neurons that have not contacted the correct targets and/or are late arriving, and subsequently, have not sequestered sufficient levels of NGF. Any basal TrkA activation in these neurons would serve to undermine the biological purpose of the cell death period, which is to ensure that only those neurons that are appropriately connected are maintained. Moreover, even in neurons that have sequestered limited NGF, SHP-1 regulation of TrkA signaling may well serve to regulate the precise balance between “positive” TrkA and “negative” p75NTR
signaling, a balance that is essential for establishment of appropriate neuron numbers.
Until recently, SHP-1 expression was largely thought to be restricted to the hematopoietic system. However, recent studies have demonstrated that SHP-1 is expressed throughout the central nervous system in both neurons (Jena et al., 1997
; Horvat et al., 2001
) and glia (Massa et al., 2000
). SHP-1 plays a key role in oligodendrocyte and glial development, as me/me
mice display decreased numbers of central nervous system glia and dysmyelination (Massa et al., 2000
; Wishcamper et al., 2001
). Together, these observations suggest an important role for SHP-1 in the development and maintenance of the nervous system, a role that we propose is mediated at least partially via regulation of the TrkA neurotrophin receptor.