Tau and signal transduction

The discovery of tau by the Kirschner lab was based on its ability to associate with microtubules and to promote microtubule assembly (Weingarten et al., 1975). After the primary sequence of tau and MAP2 were reported and functional studies performed, it became clear that both proteins contained a similar microtubule binding domain in the carboxy terminal portion of the protein (Butner & Kirschner, 1991; Ennulat et al., 1989; Himmler et al., 1989; Lee et al., 1988; Lewis et al., 1988). However, most of the remaining tau sequence was distinct from that of MAP2. Given the importance of tau and its novel role in neurodegenerative disease, a natural question was the functional significance of its amino terminal domain. This motivated our lab to seek out new functions for tau that did not require or involve microtubule association.

Our lab found that in response to nerve growth factor stimulation, tau potentiated AP-1 transcription factor activation (Leugers et al., 2013; Leugers & Lee, 2010). We also determined that this effect, at least in part, was mediated by the ability of tau to potentiate the activation of MAPK. Our findings were made using PC12-derived cell lines. In addition, we ascertained that phosphomimetic mutations in tau that compromised microtubule binding (S262D/S356D) did not affect its effects on MAPK activation. Moreover, T231D, a mutation that reduced microtubule association, significantly increased the ability of tau to potentiate MAPK activation beyond the extent exhibited by wild type tau. These data suggested that independent of its microtubule binding function, tau was capable of affecting signal transduction during the early response to NGF; a similar effect was found with EGF (Leugers & Lee, 2010).

A relationship between tau and AP-1 had previously been suggested by the microarray analysis of tau knockout mice where it was reported that the genes with the highest levels of alteration were FosB and c-fos (Supplemental Data in, Oyama et al., 2004). FosB and c-fos are part of transcription factor AP-1 and have been implicated as regulators of cell proliferation and differentiation. The finding that tau knockout mice, at 8 weeks old, expressed higher levels of FosB and c-fos relative to wild type might seem to contradict our finding that a PC12-derived cell line with tau depletion had a lower level of NGF-induced AP-1 activation relative to control. However, establishing a tau depleted cell line would not face the same pressures as when establishing a tau knockout mouse where mouse viability and ability to breed would be critical. We speculate that the undifferentiated tau depleted cell line did not need to up-regulate fosB/c-fos to proliferate, but once NGF was added, defects in MAPK and AP-1 activation were evident. An important question is by what mechanism did tau promote activation of AP-1 and MAPK?

When one considers tau's ability to influence signal transduction, there are a number of possibly relevant tau interactors that have been identified. We reported an interaction between tau and src-family non-receptor tyrosine kinases that was mediated by the SH3 domain of Src or Fyn and the proline-rich region of tau just upstream of the microtubule binding domain (Lee et al., 1998). Identification of the specific prolines involved in the interaction has been reported (Lau et al., 2016; Reynolds et al., 2008). However, the likelihood of Fyn or Src being involved in tau's effect on MAPK is low since both the Fyn and Src SH3-tau interactions were reduced by at least 8-fold when the T231D/S235D mutation was tested using surface plasmon resonance (Bhaskar et al., 2005). It is likely that the T231D mutation was solely responsible since the testing of S235E later showed no effect on the ability of tau to bind Fyn SH3 (Reynolds et al., 2008). On the other hand, we have identified an interaction between tau and protein tyrosine phosphatase SHP2 and have found that the association, as determined by proximity ligation assays, was significantly increased in PC12-like cells responding to NGF; complexes between tau and activated SHP2 were also detected (Kim et al., 2019). Moreover, in PC12-derived cells responding to NGF, phospho-T231-tau-SHP2 complexes were found, consistent with the finding that a significant increase in phospho-T231-tau occurred after 5 min of NGF stimulation. In comparing T231D against the T231A construct, significantly more T231D-SHP2 complexes occurred relative to T231A-SHP2 complexes, suggesting that phosphorylation at T231 increased SHP2 interaction. Tau-SHP2 complexes were also found in tau transfected COS cells, where EGF stimulation increased tau-activated SHP2 complexes and localized them to membrane ruffles (Kim et al., 2019). Given that SHP2 participates in the early NGF and EGF responses of PC12 cells and is required for MAPK activation and neurite outgrowth (D'Alessio et al., 2007; Okada et al., 1996; Shi et al., 2000; Wright et al., 1997), the possibility that the tau-SHP2 interaction has a role in MAPK activation needs to be investigated.

Another tau interactor that may have a role in AP-1 activation is phosphatidylinositol 3-kinase (PI3K). Tau has been shown to interact with the SH3 domain of the p85 subunit of PI3K (Reynolds et al., 2008) and while the function of the interaction remains unclear, PI3K is activated by NGF in PC12 cells (Ashcroft et al., 1999) and the ability of PI3K to activate AP-1 has been reported (Fleegal & Sumners, 2003). Interestingly, using prostate cancer cells, co-immunoprecipitates of tau and PI3K have been found (Souter & Lee, 2009) and such complexes might also exist in PC12 cells. The role of tau in non-neuronal cancer cells, where cell cycle regulation has been altered, may have relevance toward a role for tau in EGF-induced proliferation. In addition, when one considers growth factor signaling mechanisms, upstream of PI3K, Ras, and SHP2 is the grb2-SOS complex (reviewed in Belov & Mohammadi, 2012; Mendoza et al., 2011; Schlessinger, 1994; Tari & Lopez-Berestein, 2001) and, strikingly, tau also interacts with one of the SH3 domains of grb2 (Reynolds et al., 2008). Grb2 is an adapter protein that is critical for many signaling pathways and mice that are grb2 deficient are embryonic lethal (Cheng et al., 1998). It would be vital to determine the functional significance of its interaction with tau.

An additional tau interactor to consider is phospholipase Cγ1 (PLCγ1). Tau antibodies co-immunoprecipitated PLCγ (Jenkins & Johnson, 1998) and in vitro, tau interacted with the SH3 domain of PLCγ1 (Reynolds et al., 2008). Since the activation of PLCγ1 occurs following NGF treatment of PC12 cells and is essential for MAPK activation (Rong et al., 2003; Stephens et al., 1994), the tau-PLCγ1 interaction might have a role in MAPK activation. Lastly, additional tau interactors with known roles in signal transduction, such as Abl and 14-3-3, have been identified (Agarwal-Mawal et al., 2003; Derkinderen et al., 2005; Hashiguchi et al., 2000). Abl and 14-3-3 both participate in the NGF response of PC12 cells, so there is no shortage of mechanisms to consider.

Given the number of signal transduction related tau interactors that have been reported, much remains to be done. In particular, besides assuring that the associations occur in cells, as has been demonstrated for Fyn, SHP2, PI3K, Syk, and PLCγ (Jenkins & Johnson, 1998; Kim et al., 2019; Lebouvier et al., 2008; Lee et al., 1998; Souter & Lee, 2009), it would be important to determine if the association affects the function of either the interactor or tau. Tau has been established as a substrate for tyrosine kinases (Fyn, Src, Syk, Lck, and Abl; (Derkinderen et al., 2005; Lebouvier et al., 2008; Lee et al., 1998; Lee et al., 2004; Scales et al., 2011), unpublished data) which strongly suggests that tau is engaged in signal transduction pathways. Tyrosine phosphorylated tau is also a substrate for SHP2 (Kim et al., 2019). In regards to the interactors, tau together with arachidonic acid, has been shown to activate PLCγ1 (Hwang et al., 1996) and tau has also been shown to increase the in vitro activity of Fyn as well as the activity of Src in transfected cells (Sharma et al., 2007). An additional issue that also needs more investigation is the effect of tau phosphorylation on interactions. For instance, for PI3K or PLCγ1, while the interactions between tau and their SH3 domains were decreased by phosphomimetic mutations in tau, the tau construct tested contained several phosphomimetic mutations to more precisely identify the critical site(s) (Reynolds et al., 2008); therefore, more tests are needed.

In envisioning that tau has a role in signal transduction, it is relevant to mention that the association of the amino terminus of tau with the plasma membrane of neuronal cells has been reported (Brandt et al., 1995), suggesting the association of tau with a subdomain of the plasma membrane, namely lipid rafts. Lipid rafts contain receptors that initiate signal transduction in response to hormones and growth factors and lipid rafts are thought to be critical toward a cell's response to extracellular signals (reviewed in Paratcha & Ibanez, 2002; Simons & Toomre, 2000; Tsui-Pierchala et al., 2002). Moreover, tau has been found in lipid rafts, where its presence in lipid rafts correlated with cellular responses (Hernandez et al., 2009; Kawarabayashi et al., 2004; Klein et al., 2002). An investigation of tau's role in signal transduction might involve its localization in lipid rafts.

Lastly, the ability of tau to interact with a number of tyrosine kinases and other proteins involved in signal transduction is likely to stem from its proline-rich domain, given that SH3 domains interact with PXXP motifs and tau contains seven PXXP motifs. With current advances in protein structure determination, the structure of tau in filaments and the model of tau-microtubule interactions have been reported using cryo-electron microscopy (Fitzpatrick et al., 2017; Kellogg et al., 2018; Zhang et al., 2019). However, the reported structures focused on the microtubule binding domain and did not comment on the structure of the proline-rich domain. It is not clear if investigators have ever crystallized the amino terminal domain (amino acids 1–241), given the importance of the microtubule binding domain in neurodegenerative diseases. The tau-microtubule binding model of Kellogg et al has shown that the microtubule binding repeat region of tau stretches along the surface of the microtubule (Kellogg et al., 2018), with no indication that the amino terminal domain folds back toward the microtubule binding domain. However, this does not rule out the possibility that when tau is detached from microtubules, an interactor might be capable of interacting with both the proline-rich domain and the basic microtubule binding domain of tau. In addition, when the Thr231 residue on tau is phosphorylated, it can occur as either cis-pT231 or trans-pT231 (Nakamura et al., 2012), indicating that tau structure at that part of the proline-rich domain closest to the microtubule binding domain can occur in two conformations, with non-pT231 tau contributing a third conformation. It will certainly be interesting to determine how tau structure is altered by phosphorylation of T231 and how interactions with proteins such as Fyn and SHP2 are affected by changes in the structure of the proline-rich domain caused by phosphorylation.

As we reflect on 50 years of tau research, while many questions have been answered, many more have been raised. The presence of abnormal tau in Alzheimer's disease, frontotemporal dementia, epilepsy, traumatic brain injury, and other neurodegenerative diseases has led to many investigations into the properties of abnormal tau, meaning tau isolated from diseased brains, abnormally phosphorylated tau, or tau bearing FTDP-17 mutations. However, the function of normal tau, beyond its ability to promote microtubule assembly, remains to be fully established. Here we have focused on just one possible avenue for investigation, with the thought that identifying new mechanisms through which tau acts would serve as natural starting points for disease related studies. During the next 50 years, we expect tau research to elucidate new functions for tau that will lead to new disease therapies.