Phosphorylation and dephosphorylation have proved to be among the most versatile and functionally important types of posttranslational modifications (Manning et al., 2002
; Alonso et al., 2004
). In the case of NG2-dependent mechanisms, PKCα-mediated phosphorylation of NG2 at Thr2256
triggers increased cell motility via a mechanism that involves the translocation of NG2 to sites in leading edge lamellipodia of motile cells (Makagiansar et al., 2004
). This profound effect of phosphorylation on NG2 localization/function prompted us to investigate the existence of additional NG2 phosphorylation sites capable of influencing other aspects of cell biology.
Sequence analysis allows us to identify two previously unrecognized features of the NG2 cytoplasmic domain: (1) a putative D domain–docking site for ERK (residues 2,278–2,290) and (2) a potential site for ERK-mediated phosphorylation at Thr2314. Immunoprecipitation/immunoblotting results confirm the existence of a physical interaction between NG2 and activated ERK. In addition, purified recombinant ERK is able to phosphorylate the isolated NG2 cytoplasmic domain at Thr2265 and Thr2314.
In extending these studies to full-length NG2 expressed endogenously by A375 melanoma cells or by transfection in U251 glioma cells, we used PDGF-BB stimulation to activate both PKCα and ERK. This provides a more physiologically relevant means of stimulating phosphorylation than the PMA treatment we previously used to identify PKCα-mediated modification at Thr2256
(Makagiansar et al., 2004
). We evaluated PDGF-BB–induced phosphorylation of NG2 by immunoblotting with phosphospecific antibodies against the xTpx(K/R) motif present at Thr2256
and against the xTpP motif present at Thr2314
. The use of specific inhibitors of PKCα and ERK coupled with analysis of NG2 species with valine substitutions at the putative phosphorylation sites reveals that PKCα-mediated phosphorylation occurs at Thr2256
, whereas ERK-mediated phosphorylation occurs only at Thr2314
. Contrary to the results obtained in vitro with recombinant ERK treatment of the isolated cytoplasmic domain, Thr2265
is not used as a site for ERK phosphorylation in full-length NG2 expressed by living cells. This is more in keeping with predictions based on NG2 sequence analysis because the residues in the immediate vicinity of Thr2265
do not represent a canonical ERK phosphorylation motif.
An important mechanistic observation from the experiments with NG2-transfected U251 cells is that ERK activation in response to PDGF-BB is highly dependent on the activity of PKCα. This seems somewhat surprising because conventional wisdom would suggest that PDGF receptors should also be able to activate ERK via a Son of sevenless–Ras–Raf–MEK-dependent pathway (for review see Porter and Vaillancourt, 1998
). However, the ability of the PKCα inhibitor Gö6976 to inhibit both the PKCα-catalyzed phosphorylation of Thr2256
and the ERK-catalyzed phosphorylation of Thr2314
does not provide evidence for an important contribution of the growth factor–activated Son of sevenless–Ras pathway to ERK activation in U251 cells. A similar dependence of ERK on PKC activity has been observed in PDGF stimulation of smooth muscle cells (Robin et al., 2004
; Ginnan and Singer, 2005
). The consequences of these findings in U251 cells are twofold: (1) PDGF-BB treatment inevitably activates both PKCα and ERK, thus leading to NG2 phosphorylation at both Thr2256
, and (2) we cannot inhibit PKCα without also affecting ERK activity. In this regard, the constitutively active MEK-DD construct has provided an effective means for activating ERK independent of PKCα activation, enabling us to restrict NG2 phosphorylation to Thr2314
Having established the existence of two distinct types of Thr phosphorylation sites in the NG2 cytoplasmic domain, we addressed the effects of these two phosphorylation events on cell behavior. Use of the transwell cell motility assay allows us to confirm our previous report (Makagiansar et al., 2004
) that both the actual and mimicked phosphorylation of NG2 at Thr2256
leads to enhanced cell migration. Not surprisingly, PDGF-BB increases the motility of parental U251 cells via mechanisms that are independent of NG2. Nevertheless, the presence of NG2 further enhances the response to PDGF-BB. The fact that this is caused by NG2 phosphorylation at Thr2256
is demonstrated by the loss of enhanced PDGF-BB responsiveness in NG2-T2256V transfectants and by the increased motility of NG2-T2256E variants even in the absence of growth factor. Notably, the other NG2 transfectant that fails to exhibit enhanced NG2-dependent motility in response to PDGF-BB is the NG2-T2314E variant. Because the NG2-T2314V variant does not exhibit this loss of function, the depressed motility of NG2-T2314E transfectants cannot be caused by the blockage of Thr2314
phosphorylation. Instead, the defect must be the result of mimicked phosphorylation at this site, indicating that phosphorylation at Thr2314
counteracts the stimulation of motility produced by the phosphorylation of Thr2256
We were also able to identify effects of NG2 phosphorylation on cell proliferation. In the absence of growth factor stimulation, U251 cells expressing NG2 exhibit a greater rate of cell proliferation than parental U251 cells. This phenomenon is observed in both conventionally transfected and adenovirally transformed cells, demonstrating that the behavior is independent of the means of NG2 expression. Elevated basal rates of proliferation are also observed in each of the valine-substituted NG2 variants with the exception of NG2-T2314V, suggesting that basal levels of phosphorylation at Thr2314 in nonstimulated cells () may be responsible for the increased proliferation of NG2-expressing U251 cells relative to parental cells. Support for this idea is provided by the behavior of U251/NG2/MEK-DD transfectants and NG2-T2314E transfectants, both of which proliferate faster under basal conditions than any other species examined because of phosphorylation and mimicked phosphorylation at Thr2314, respectively.
Interestingly, although the addition of PDGF-BB has no effect on the proliferation of parental U251 cells, it negatively affects the proliferation of U251/NG2 transfectants. This negative effect of PDGF is also seen in the valine-substituted NG2 variants with the exception of NG2-T2256V, in which proliferation is enhanced. The possibility that PDGF-induced phosphorylation at Thr2256 is capable of reversing the stimulatory effect on proliferation caused by Thr2314 phosphorylation is supported by the low rates of proliferation observed in NG2-T2256E transfectants, which mimic Thr2256 phosphorylation.
Our findings suggest the existence of an intriguing balance between cell proliferation and migration that can be regulated, in part, by phosphorylation of the NG2 cytoplasmic domain at two different sites. PKCα-mediated phosphorylation at Thr2256 appears to stimulate cell motility while inhibiting cell proliferation. Conversely, ERK-mediated phosphorylation at Thr2314 tends to block cell motility while promoting cell proliferation. In unstimulated cells, it appears that low levels of Thr2314 phosphorylation are responsible for the increased rate of proliferation of U251/NG2 cells compared with parental U251 cells. When cells are treated with PDGF-BB, increased phosphorylation occurs at both Thr2314 and Thr2256. Under these conditions, our data suggest that signals generated via Thr2256 phosphorylation may be dominant over those resulting from Thr2314 phosphorylation.
In this context, and considering that both PDGF-induced phosphorylation events seem to depend on PKCα activation, it is logical to ask under what circumstances Thr2314
phosphorylation would lead to increased cell proliferation. An answer may lie in the ability of non–growth factor–driven mechanisms to activate ERK independently of PKCα activation. An artificial example of such a mechanism is provided by our use of the MEK-DD construct to drive ERK activation independently of PKCα. Under these circumstances, Thr2314
phosphorylation is achieved in the absence of Thr2256
phosphorylation, resulting in the enhancement of cell proliferation. A more biologically relevant means of achieving Thr2314
phosphorylation independent of Thr2256
phosphorylation might involve the activation of ERK via a G protein–coupled receptor-dependent pathway (Pierce et al., 2001
). Alternatively, integrin-mediated activation of the FAK–src–p130cas pathway could serve to stimulate ERK independently of PKCα (Defillippi et al., 2006
With respect to integrins, it is noteworthy that we and others have reported the ability of NG2 to activate β1-integrin signaling (Iida et al., 1995
; Fukushi et al., 2004
; Yang et al., 2004
). NG2 is able to form a signaling complex with α3β1-integrin when the proteoglycan is present in soluble exogenous form or when it is expressed in cis fashion on the same cells as the integrin (Fukushi et al., 2004
). The relationship between NG2 and β1-integrins may be especially relevant to the behavior of U251/NG2 cells. Labeling with the activation-dependent HUTS-21 antibody reveals β1-integrin activation in U251 cells that express the NG2-T2256E and NG2-T2314E variants that mimic phosphorylation at Thr2256
, respectively. In addition, a β1-blocking antibody has inhibitory effects on both the proliferation and motility induced by NG2 phosphorylation at Thr2314
These results are initially paradoxical because it is not immediately clear how integrin activation by NG2 might be able to stimulate proliferation in one case (response to Thr2314
phosphorylation) and motility in the other (response to Thr2256
phosphorylation). We suggest two possible resolutions to this paradox. First, the two phosphorylated NG2 species might differentially influence integrin signaling by interacting with different β1-integrin heterodimers or by recruiting additional distinct cytoplasmic binding partners to the NG2–β1-integrin complex. Second, differential localization of the NG2–integrin complexes may be determined by the NG2 phosphorylation pattern with the result that integrin signaling occurs in distinctly different microdomains of the cell. We have presented evidence consistent with this second alternative. Specifically, the NG2-T2256E species is localized along with β1-integrin in broad lamellipodia so that integrin activation and the resulting tyrosine phosphorylation of downstream signaling intermediates are localized to a microdomain that is critical to cell motility. In contrast, the NG2-T2314E species is colocalized with β1-integrin and elevated tyrosine phosphorylation on apical microprotrusions that appear to be dependent on NG2 phosphorylation at Thr2314
for their formation. These observations are consistent with the concept that integrin-mediated signal transduction can activate both motility and proliferation via intermediates such as FAK and Crk-associated substrate, whose specific localization patterns determine the outcome of signaling (for review see Cox et al., 2006
These proliferation and motility results obtained with NG2-transfected U251 cells contradict, to some extent, our previous results with aortic smooth muscle cells from wild-type and NG2-null mice (Grako et al., 1999
). Wild-type smooth muscle cells proliferated and migrated in response to both PDGF-AA and -BB. NG2-null cells failed to respond well to PDGF-AA but had normal responses to PDGF-BB. Thus, unlike our results with U251 glioma cells, the presence or absence of NG2 did not affect smooth muscle cell responses to PDGF-BB. It seems likely to us that these apparent discrepancies reflect the primitive status of our understanding of differences in the details of signaling mechanisms that occur from one cell type to another. Several examples will serve to illustrate the complexity of the situation. (1) We have no information about the phosphorylation of NG2 in smooth muscle cells. If PKCα and ERK are not activated by PDGF-BB in these cells in the same way we have seen in U251 and A375 cells, the phosphorylation of NG2 at Thr2256
will not occur, and NG2 will have no influence on motility or proliferation. (2) As a result of the proposed link between integrin signaling and PDGF receptor activation (Borges et al., 2000
), the NG2-dependent integrin activation described in this study could have an effect on PDGF receptor signaling. Because we have not determined how the spectrum of integrins in smooth muscle cells compares with that of U251 cells, we cannot say whether integrin–PDGF receptor or NG2–integrin interactions would be comparable between the two cell types. (3) PDGF-BB activates signaling through both α and β receptors. We have presented evidence for an interaction between NG2 and PDGFα receptor that potentiates signaling via this receptor (Nishiyama et al., 1996
; Grako et al., 1999
). The relative abundance of α and β receptors has been found to differ between smooth muscle and glioma cells (Sachinidis et al., 1990
; Lokker et al., 2002
), and we have not determined the α/β ratio in the specific cases of our two cell types. Therefore, we cannot predict the extent to which NG2 itself affects PDGF receptor signaling in either cell type. These uncertainties make it clear that our understanding of the functional role of NG2 is at an early stage and that much additional work will be required to elucidate details of the mechanisms by which NG2–integrin–PDGF receptor interactions regulate cell proliferation and motility.