Distinct Priming Kinases Contribute to Differential Regulation of Collapsin Response Mediator Proteins by Glycogen Synthase Kinase-3 in Vivo

Abstract

Collapsin response mediator proteins (CRMPs) are a family of neuron-enriched proteins that regulate neurite outgrowth and growth cone dynamics. Here, we show that Cdk5 phosphorylates CRMP1, CRMP2, and CRMP4, priming for subsequent phosphorylation by GSK3 in vitro. In contrast, DYRK2 phosphorylates and primes CRMP4 only. The Cdk5 and DYRK2 inhibitor purvalanol decreases the phosphorylation of CRMP proteins in neurons, whereas CRMP1 and CRMP2, but not CRMP4, phosphorylation is decreased in Cdk5 ؊/؊ cortices. Stimulation of neuroblastoma cells with IGF1 or TPA decreases GSK3 activity concomitantly with CRMP2 and CRMP4 phosphorylation. Conversely, increased GSK3 activity is not sufficient to increase CRMP phosphorylation. However, the growth cone collapse-inducing protein Sema3A increases Cdk5 activity and promotes phosphorylation of CRMP2 (but not CRMP4). Therefore, inhibition of GSK3 alters phosphorylation of all CRMP isoforms; however, individual isoforms can be differentially regulated by their respective priming kinase. This is the first GSK3 substrate found to be regulated in this manner and may explain the hyperphosphorylation of CRMP2 observed in Alzheimer's disease.Glycogen synthase kinase 3 (GSK3) 4 is an evolutionarily conserved and ubiquitously expressed Ser/Thr kinase that is expressed as two closely related isoforms in mammals, GSK3␣ (51 kDa) and GSK3␤ (47 kDa) (1). GSK3 is unusual when compared with other protein kinases as it is constitutively active in cells and phosphorylation of most substrates must be preceded by phosphorylation of a nearby residue by another kinase. This process is known as priming and occurs at Ser/Thr residues located 4 or 5 residues C-terminal to the site phosphorylated by GSK3 (2, 3). GSK3 activity is inhibited by phosphorylation of an N-terminal serine residue (Ser 21 on GSK3␣ and Ser 9 on GSK3␤), which is catalyzed by members of the AGC family of protein kinases upon stimulation by growth factors (4, 5). Alternatively, GSK3 activity may be inhibited by protein-protein interactions following activation of the Wnt signaling pathway (6, 7). It is also possible that regulation of priming kinases could indirectly regulate phosphorylation of substrates by GSK3, although this has yet to be proven.We have recently discovered new brain-specific substrates for GSK3, namely collapsin response mediator protein (CRMP) 2 and 4 (3). These isoforms are members of a family of five CRMP proteins (CRMP1-5) that are expressed almost ubiquitously throughout the central nervous system (8, 9). CRMP2 is the best studied isoform of the family. Mammalian CRMP2 binds to tubulin heterodimers to promote microtubule formation and co-localizes with microtubules inside cells (10). Overexpression of CRMP2 in hippocampal neurons promotes increased axon elongation (3, 10, 11). However, mutation of the GSK3 phosphorylation sites on CRMP2 to non-phosphorylatable alanine residues alters CRMP2-induced axon elongation (3, 12). Other functions attributed to CRMP2 include regulation of cell surface receptor intern...

Probing the lithium-response pathway in hiPSCs implicates the phosphoregulatory set-point for a cytoskeletal modulator in bipolar pathogenesis

“…1B). Interestingly, inhibitors of CDK5 (cyclin-dependent kinase-5), the presumed S522 kinase that primes other CRMP2 sites for subsequent GSK3β phosphorylation (25), did not decrease CRMP2-p-T514, suggesting that lithium decreases CRMP2-p-S522 independently of CDK5 in human neurons (SI Appendix, Fig. S9C).…”

“…However, phosphorylation of CRMP2-T514 (as well as of CRMP2-S518 and CRMP2-T509) must first be primed by phosphorylation at CRMP2-S522. Phosphorylation of CRMP2 by kinases is balanced by its dephosphorylation by phosphatases (24)(25)(26) (Fig. 1B).…”

“…It is known that phosphorylation of CRMP2 at threonine (T) 514 (CRMP2-p-T514) causes its dissociation from cytoskeletal proteins (e.g., tubulin heterodimers) (24)(25)(26). However, phosphorylation of CRMP2-T514 (as well as of CRMP2-S518 and CRMP2-T509) must first be primed by phosphorylation at CRMP2-S522.…”

Cdk5‐mediated phosphorylation of CRMP‐2 enhances its interaction with CaV2.2

“…Based upon the established important role of phosphorylation in modulating CRMP-2's activity and interaction with partners proteins, we hypothesized that phosphorylation may also affect its modulation of CaV2.2. Here, we focused solely on evaluating the effect of Cdk5-phosphoryalted CRMP-2 on CaV2.2 because (i) Cdk5 is an obligatory priming kinase [28], (ii) Cdk5-phosphorylated CRMP-2 (at Ser-522) has reduced interaction with partner proteins tubulin and numb [24], (iii) Cdk5-phosphorylated CRMP-2 leads to altered neuronal functionality manifested as a halt in neurite growth [29], and (iv) the Cdk5-phosphorylation site is near the CBD3 binding site on CRMP-2 that is important for its interaction with CaV2.2. Our results show that mutation of the Cdk5-phosphorylation site, but not the RhoK phosphorylation site, regulates CRMP-2-mediated enhancement of Ca 2+ influx.…”

Hierarchical CRMP2 posttranslational modifications control NaV1.7 function

“…CRMP2 regulates multiple processes in neurons and was initially discovered to regulate mechanisms of neuronal polarity (9,10). CRMP2 phosphorylation by cyclin-dependent kinase 5 (Cdk5) (11), glycogen synthase kinase 3β (10), Rho-associated protein kinase (12), or the Src-family kinases Fyn (13) and Yes (14) drives its diverse cellular functions, including neurite outgrowth, endocytosis, and ion-channel trafficking (8,(15)(16)(17). Studies of CRMP2 trafficking functions have revealed that CRMP2 facilitates endocytosis of L1-cell adhesion molecule by interacting with the endocytic protein Numb (18) that recruits epidermal growth factor receptor pathway substrate 15 (Eps15), an initiator of clathrin-mediated endocytosis (19).…”