MAP2 was identified as a microtubule-associated protein due to its ability to bind to and stabilize microtubules. It exerts this stabilization effect by reducing the frequency of catastrophes and slowing down the shortening rate of microtubules (Gamblin et al., 1996; Itoh and Hotani, 1994). Furthermore, MAP2 can facilitate microtubule bundling, acting as a spacer within the microtubule bundle (Chen et al., 1992; Weisshaar and Matus, 1993). MAP2 has been shown to promote tubulin polymerization through its microtubulin-binding region, which can be regulated by phosphorylation (Ainsztein and Purich, 1994). MAP2, via its microtubulin-binding region can also bind to other cytoskeleton components, such as actin, acting as the crosslinker between microtubule and actin. MAP2 can also interact with Plectin (an intermediate filament-associated cytolinker protein), which can antagonize the microtubule stabilization ability of MAP2 (Valencia et al., 2013).

Neurite initiation depends on the rapid reorganization of the cytoskeleton, achieved through the coordination of microtubules and actin filaments. Given its ability to influence both microtubule and actin organization, it creates a conducive environment for triggering neurite initiation (L. Dehmelt et al., 2003). MAP2 has been shown to be shifted and concentrated in the outgrowth and branching areas of dendrites in several cell lines (Matus et al., 1986; Ferreira et al., 1989; Chamak et al., 1987). Evidence indicates that the expression of MAP2c can induce the rapid formation of dense and stable microtubule bundles at the distal plus end. Conversely, knocking down MAP2 in primary cortical neurons and hippocampal neurons results in a reduction in both the number and length of dendrites, as well as a decrease in microtubule presence within dendrites (Sharma et al., 1994; Harada et al., 2002; LeClerc et al., 1993; Dehmelt et al., 2003). MAP2 serves as the receptor for neurosteroid pregnenolone (PREG) to enhance neurite outgrowth by stimulating microtubule polymerization (Fontaine-Lenoir et al., 2006).

MAP2 is essential for long-term potentiation (LTP) and the dendritic spine changes induced by LTP. Kim et al. have demonstrated that MAP2 is translocated to the spines in response to LTP stimulation and the knockdown of HMW MAP2 causes a deficit of LTP induction and abolishes LTP-induced surface delivery of AMPA receptors and spine enlargement. However, the knockdown of LMW MAP2 does not show the same effect (Kim et al., 2020). Also, MAP2 contributes to the inhibition of microtubule entry into dendritic spines induced by long-term depression (LTD) by mediating the prolonged redistribution of EB3 (End-binding protein 3) along MAP2-positive microtubule bundles in the dendritic shaft, thereby suppressing microtubule dynamics (Kapitein et al., 2011). EB3 is a member of microtubule plus-end binding proteins, which regulate microtubule dynamics (Jaworski et al., 2009).

MAP2 has been reported to show a chaperone-like activity, preventing protein aggregation under thermal or chemical induction and assisting in enzyme refolding (Sarkar et al., 2004). Specifically, MAP2c plays a role in preventing tau aggregation, which is implicated in Alzheimer’s disease. Electron microscopy studies show that MAP2c inhibits arachidonic acid-induced tau aggregation in vitro, suggesting a role in maintaining tau homeostasis. Despite the high homology between the C-terminal regions of tau and MAP2c, the N-terminal region of MAP2c alone does not mediate its chaperone activity (Mitra et al., 2015).

MAP2 is responsible for regulating kinesin/dynein-dependent microtubule transporting in neurons (Heins et al., 1991; Lopez and Sheetz, 1993; Seitz et al., 2002; Hagiwara et al., 1994). This regulation is achieved in two ways: (1) MAP2 and motor proteins both bind to the C-terminus of tubulin, competing for the same binding sites on microtubules; (2) HMW MAP2 can interact with motor proteins, like KIF5B, preventing its binding to microtubules through the steric inhibition caused by MAP2B’s projection domain. By coordinating with motor proteins, MAP2B forms a filtering zone at the axon initial segment that controls cargo entrance to the axon in sensory neurons (Hagiwara et al., 1994; Gumy et al., 2017). The regulation by MAP2 of cargo transportation thus enables the polarized distribution of neuronal proteins and organelles, which is vital to normal neuronal function.

MAP2 serves as the dominant anchoring protein of cAMP-dependent protein kinase (PKA) in dendrites, whose kinase activity is known to be involved in various biological functions in neurons (Abel et al., 1997; Malleret et al., 2001). MAP2 binds to the regulatory subunit II (RII) of PKA through its N-terminus and determines the localization of PKA in neurons (Vallee et al., 1981; Theurkauf and Vallee, 1982). MAP2 serves as a reservoir for PKA. A deficit in MAP2 impairs the ability of PKA to phosphorylate key substrates, such as CREB and AMPA receptors, following signal stimulation. This disruption ultimately blocks the downstream signal transduction pathway (Zhong et al., 2009; Harada et al., 2002). Furthermore, the RII subunit of PKA, but not RI, binds specifically to MAP2 and is shielded from calpain degradation when associated with MAP2 (Alexa et al., 1996).

Additionally, MAP2 comprises 11 PXXP motifs which are the potential binding ligands of Src homology 3 (SH3) domains. MAP2 interacts with several SH3 domain-containing proteins, for example, the non-receptor protein tyrosine kinase Src and Fyn and the adaptor protein Grb2 (see 4.1 below). Src family kinases are a group of non-receptor kinases containing conserved SH3 and SH2 domains, which mediate substrate recognition and protein–protein interactions (Parsons and Parsons, 2004). These kinases play critical roles in neuronal signaling and cytoskeletal regulation. Also, the MAP2 Co-IP experiment identified several SH-domain-containing proteins within the MAP2 interactome, including guanine nucleotide exchange factors and members of the Rho family of GTPases, both of which are involved in signal transduction (Lyu et al., 2024). The interaction between MAP2 and proteins mentioned above may indicate the role of MAP2 as a scaffold protein.

MAP2 has been implicated in the regulation of protein synthesis. Overexpression of the MAP2c isoform has been shown to inhibit protein synthesis in HEK cells, suggesting a direct role in translational control, though the mechanism is still unclear (Grubisha et al., 2021). This inhibitory role is further supported by the composition of the MAP2 interactome, which includes multiple RNA-binding proteins known to modulate translation, many of which are associated with suppressing protein synthesis. A key interaction underlying this regulatory function is between MAP2 and insulin-like growth factor 2 mRNA-binding protein 1 (IMP1). MAP2 binds directly to IMP1 via its K-homology (KH) domains, which are critical for nucleic acid binding (Nielsen et al., 2002). Furthermore, proteomic analyses of the MAP2 interactome have identified numerous RNA-binding proteins involved in the regulation of translation, many of which are associated with inhibitory effects on protein synthesis (Grubisha et al., 2021). Together, these findings suggest that MAP2 contributes to the modulation of protein synthesis, potentially through interactions with RNA-binding proteins and direct effects on translational machinery.

MAP2 contains a proline-rich RTPPKSP motif which specifically interacts with the SH3 domain of Src family kinases. Among the Src family kinases, MAP2 has been reported to interact with two key members of the Src family kinase, Fyn and Src (Zamora-Leon et al., 2001; Sontag et al., 2012). Phosphorylation of MAP2 by Fyn was initially demonstrated in vitro (Zamora-Leon et al., 2001), with mutagenesis studies of MAP2c later identifying tyrosine 67 (Y67) as the primary phosphorylation site on the MAP2c isoform (Zamora-Leon et al., 2005). Phosphorylation at this site has also been confirmed in the human fetal brain, and it assists in recruiting the SH2 domain of Grb2 (Zamora-Leon et al., 2005). The binding of Fyn to MAP2 also inhibits the interaction between MAP2 and protein phosphatase 2A (PP2A), thereby suppressing PP2A’s dephosphorylation activity, which may stabilize MAP2 phosphorylation states (Sontag et al., 2012).

Interestingly, although MAP2 interacts with Src via the SH3 domain, Src does not phosphorylate MAP2 in vivo (Lim and Halpain, 2000).

PDPKs are a class of serine/threonine kinases that specifically phosphorylate substrates at serine or threonine residues followed by a proline (Ser/Thr-Pro motifs). These kinases are crucial regulators of cell signaling pathways. The MAPK (mitogen-activated protein kinase) family, cyclin-dependent kinases (CDKs), and glycogen synthase kinase 3 (GSK-3) are among the well-known PDPKs. MAP2’s proline-rich domain is abundant in Ser/Thr-Pro motifs, making it a target for PDPKs.

Microtubule affinity-regulating kinases (MARKs), also known as Par-1 kinases, are serine/threonine kinases that regulate microtubule dynamics by phosphorylating microtubule-associated proteins (MAPs), including MAP2, MAP4, and tau (Drewes et al., 1997). MARKs are involved in maintaining cytoskeletal stability, neuronal polarity, and intracellular transport, making them essential for proper neuronal function (Drewes et al., 1997).

Microtubule affinity-regulating kinases (MARKs) were first identified for their ability to phosphorylate microtubule-associated proteins (MAPs) and disrupt their interaction with microtubules (MTs), profoundly altering cytoskeletal dynamics (Drewes et al., 1995). The first discovered MARK isoform, p110^MARK, was initially characterized for phosphorylating tau and later shown to phosphorylate MAP2 at conserved KXGS motifs in the MAP2 MT binding domain (Drewes et al., 1995). Two major phosphorylation sites in MAP2 targeted by p110^MARK are Ser1713 (KCGS motif) and Ser1682 (KIGS motif). This phosphorylation dissociates MAP2 from MTs, leading to increased dynamic instability of the microtubule network (Illenberger et al., 1996).

Further studies demonstrated that MARK1 and MARK2, primarily expressed in brain, share this ability to efficiently block MAP2-MT interactions (Drewes et al., 1997). Overexpression of MARK1 or MARK2 in CHO cells results in the destruction of the microtubule network without affecting the microfilament network, highlighting their specific regulatory role. Conversely, co-transfection with a MAP2c mutant (S319A/S350A) resistant to MARK phosphorylation effectively counteracted this microtubule destabilization, underscoring the direct correlation between KXGS motif phosphorylation and cytoskeletal disassembly (Drewes et al., 1997).

PKA, a serine/threonine kinase activated by cyclic AMP (cAMP), plays a critical role in regulating cytoskeletal dynamics and synaptic plasticity (Huang et al., 2013). MAP2 serves as a major substrate of PKA in neurons (Theurkauf and Vallee, 1982, 1983). The regulatory subunit RII of PKA interacts with a conserved 83-amino-acid sequence at the distal N-terminus of MAP2 isoforms (MAP2A, MAP2B, and MAP2C) (Obar et al., 1989). In vitro studies demonstrate that PKA can phosphorylate MAP2B at up to 15 moles of phosphate per molecule, with 70% of these phosphorylation events distributed across the proline-rich and microtubule-binding domains (Itoh et al., 1997). Detailed analyses using tryptic digestion and two-dimensional phosphopeptide mapping identified 11 major PKA-specific phosphorylation sites, all localized to serine residues (Goldenring et al., 1985). These phosphopeptides were also present in the rat brain and GH3 cells (Diaz-Nido et al., 1990; Jefferson and Schulman, 1991). NMR spectrometry has further resolved these phosphorylation sites on MAP2C with single-residue precision (Jansen et al., 2017). Notably, four PKA phosphorylation sites overlap with those targeted by calcium/calmodulin-dependent protein kinase II (CaMKII) (Walaas and Nairn, 1989). During development, in cytosol, PKA phosphorylation of MAP2 increased from 2 days to adult in proportion to the increase in the concentration of MAP2 in chicken brains (Koszka et al., 1991).

Phosphorylation of MAP2 by PKA alters its interactions with microtubules and other cellular structures. PKA-mediated phosphorylation of MAP2 reduces its ability to bind to and nucleate microtubules, although its ability to stabilize microtubules remains unaffected (Itoh et al., 1997). Within the microtubule-binding domain of MAP2C, PKA phosphorylates serine residues at the KXGS motifs (S319, S350, and S382). These phosphorylations result in MAP2C dissociation from microtubules and redistribution to the peripheral actin cytoskeleton (Ozer and Halpain, 2000). Additionally, phosphorylation by PKA enhances MAP2C’s affinity for 14–3-3ζ, a regulatory protein that competes with tubulin for MAP2 binding, although this interaction does not disrupt MAP2C-microtubule binding (Jansen et al., 2017). PKA-mediated phosphorylation also abolished the chaperone-like ability of MAP2c for preventing tau fibril formation induced by arachidonic acid in vitro (Mitra et al., 2015).

The functional implications of PKA-mediated phosphorylation extend beyond microtubule dynamics. For example, phosphorylation at T220 significantly inhibits the calpain-induced hydrolysis of MAP2, a protection not observed with CaMKII-mediated phosphorylation (Johnson and Foley, 1993; Alexa et al., 1996; Alexa et al., 2002). Also, phosphorylation by PKA reduced binding to SH3 domain containing proteins, like Src and Grb2 (Lim and Halpain, 2000).

MAP2 phosphorylated by PKA from the brain can be dephosphorylated by calcineurin and protein phosphatase with Km values in the range of 1–3 μM and 1.6–2.7 μM, respectively (Goto et al., 1985; Yamamoto et al., 1988). PKA signaling also mediates dendritic remodeling. Activation of D1 dopamine receptors (D1Rs), which stimulate the cAMP-PKA pathway, increases MAP2 phosphorylation and correlates with decreased dendritic extension (Song et al., 2002).

Protein kinase C (PKC) is a serine/threonine kinase widely distributed across various brain regions. It is activated by Ca²⁺ and phospholipids (Saito et al., 1988). PKC in brain tissue can be classified into three main subtypes α, β (I and II) and γ, with primary structures highly homologous and conserved. The γ subtype is localized in the brain and spinal cord uniquely (Domanska-Janik, 1996).

PKC was first identified as a kinase capable of phosphorylating MAP2 in rat brains, using two-dimensional gel electrophoresis (Rodnight et al., 1985). Subsequent studies revealed that PKC specifically phosphorylates serine residues on MAP2 and is capable of incorporating at least 15 moles of phosphate into MAP2 (Walaas and Nairn, 1989; Akiyama et al., 1986). The phosphorylation sites targeted by PKC are located within the tubulin-binding domain (S1703, S1711, and S1728 in mouse Map2b, homologous to S1702, S1710, and S1727 in human MAP2b) and the projection domain. The homologous phosphorylation sites have been validated in rat brain tissues (S1705, S1713, and S1730 in rat MAP2b) (Tsuyama et al., 1986; Ainsztein and Purich, 1994).

Functionally, PKC-mediated phosphorylation modulates the ability of MAP2 to interact with cytoskeletal components. Overall phosphorylation by PKC reduces both microtubule polymerization and actin cross-linking induced by MAP2, while phosphorylation at S1728 completely abolishes MAP2’s microtubule-binding ability (Hoshi et al., 1988; Ainsztein and Purich, 1994; Diaz-Nido et al., 1990). Furthermore, under pathological conditions such as hyperammonemia, PKC activity on MAP2 is significantly reduced, highlighting its regulatory importance in both normal and diseased states (Felipo et al., 1993).

CAMKII is a serine/threonine kinase activated upon binding of Ca²⁺/calmodulin. It is the most abundant post-synaptic domain protein. The three isoforms of CAMKII, CaMKIIβ, CAMKIIγ and CAMKIIδ, are more broadly distributed across the brain regions and cell types, whereas CaMKIIα is predominantly expressed in excitatory neurons and certain inhibitory neurons, such as Purkinje cells (Bayer et al., 1999). CAMKII is essential to synaptic plasticity, learning, and memory (Silva et al., 1992; Yasuda et al., 2022).

CaMKII phosphorylates MAP2 in the brain, incorporating up to 5 moles of phosphate into the protein (Vallano et al., 1986; Yamauchi and Fujisawa, 1982). Tryptic digestion and two-dimensional phosphopeptide mapping identified five major phosphopeptides targeted by CaMKII, four of which involve threonine residues. The remaining serine residue is shared as a phosphorylation site with PKA (Goldenring et al., 1985; Jefferson and Schulman, 1991). Interestingly, different studies using varied phosphorylation conditions and protease digestion have reported four overlapping phosphorylation sites between CaMKII and PKA (Walaas and Nairn, 1989; Jefferson and Schulman, 1991; Schulman, 1984). These phosphorylation sites identified in vitro have also been detected in rat brains and pancreatic cells (Yamamoto et al., 1983; Yamamoto et al., 1985; Krueger et al., 1997).

The functional consequences of MAP2 phosphorylation by CaMKII include inhibition of MAP2-induced microtubule assembly (Yamamoto et al., 1983). During development, MAP2 phosphorylation level decreases in proportion to the declining concentration of CaMKII in chicken brains, indicating a developmental regulation of MAP2 function by this kinase (Koszka et al., 1991). Additionally, the reversal of CaMKII-mediated phosphorylation by calcineurin and protein phosphatase C restores MAP2’s ability to promote microtubule assembly, underscoring the dynamic regulation of MAP2 phosphorylation and dephosphorylation in cellular processes (Goto et al., 1985; Yamamoto et al., 1988).