Phosphorylation of NG2 Proteoglycan by Protein Kinase C-α Regulates Polarized Membrane Distribution and Cell Motility

Abstract

Protein kinase C (PKC)-␣ phosphorylation of recombinant NG2 cytoplasmic domain and phorbol ester-induced PKC-dependent phosphorylation of full-length NG2 expressed in U251 cells are both blocked by mutation of Thr 2256 , identifying this residue as a primary phosphorylation site. In untreated U251/NG2 cells, NG2 is present along with ezrin and ␣ 3 ␤ 1 integrin in apical cell surface protrusions. Phorbol ester treatment causes redistribution of all three components to lamellipodia, accompanied by increased cell motility. U251 cells expressing NG2 with a valine substitution at position 2256 are resistant to phorbol ester treatment: NG2 remains in membrane protrusions and cell motility is unchanged. In contrast, NG2 with a glutamic acid substitution at position 2256 redistributes to lamellipodia even without phorbol ester treatment, rendering transfected U251 cells spontaneously motile. PKC-␣-mediated NG2 phosphorylation at Thr 2256 is therefore a key step for initiating cell polarization and motility.Transmembrane proteoglycans such as CD44 and syndecans make important contributions to communication between the exterior and interior of the cell (1, 2). We are elucidating specific signaling functions for NG2, a membrane-spanning chondroitin sulfate proteoglycan found on several types of immature progenitor cells and on a variety of tumor types (3). Two hallmarks of both progenitor and tumor cells are increased motility and proliferation, both of which are influenced by NG2 (4 -7).Several mechanisms have been suggested to account for the contribution of NG2 to these processes. These include sequestration of growth factors (5, 8), modulation of the activity of kringle domain proteins (9, 10) and matrix metalloproteinases (11), and interaction with other cell surface molecules or with extracellular matrix components that regulate signaling pathways involved in cell proliferation and motility (12-15). NG2 engagement has been shown to result in activation of the small GTPases cdc42 and rac (15, 16) and in ␤ 1 integrin-dependent activation of focal adhesion kinase and ERK-1/2 1 (17, 18). The fundamental importance of these pathways in cell physiology underscores the potential significance of elucidating the specific contributions of NG2 to transmembrane communication.Protein phosphorylation and dephosphorylation are recognized as critical aspects of intracellular signaling. By altering protein conformation and by creating docking sites for proteinprotein interaction, phosphorylation and dephosphorylation of cytoplasmic tyrosine, serine, and threonine residues provide an extremely versatile means of regulating signaling pathways (19,20). Although the NG2 cytoplasmic domain contains several threonine residues that might serve as phosphorylation sites (21), to date we have had no experimental verification of this type of modification in NG2 and no information about potential functional consequences. The current work sheds initial light on these questions by identifying Thr 2256 as the primary site for NG2 modification by PK...

Interaction of Syntenin-1 and the NG2 Proteoglycan in Migratory Oligodendrocyte Precursor Cells

“…Syntenin can mediate cytoskeletal dynamics at the plasma membrane; syntenin-1 overexpression induces the formation of long, branching plasma membrane extensions (14). In astrocytomas, NG2 has been compartmentalized with membrane cytoskeletal linkers such as ERM (ezrin/radixin/moesin) proteins, which interact with actin (16). The expression of ERM proteins by oligodendrocytes has not been reported.…”

A role for the NG2 proteoglycan in glioma progression

“…23 There are several cytoplasmic threonine residues, at least two of which are sites of functionally important phosphorylation. Thr-2256 is phosphorylated by PKCα, 24 while Thr-2314 is phosphorylated by ERK. 25 Although a classical PXXP SH3 binding domain is not present, the C-terminal half of the NG2 cytoplasmic tail is very rich in prolines.…”

“…Upon stimulation with PMA or PDGF, however, U251/NG2 motility increased significantly compared to that of U251 cells. Further investigation showed that both PMA and PDGF triggered PKCα-dependent phosphorylation of NG2 at Thr-2256, 24,25 and that this phosphorylation event was required for the increase in motility. A Thr-2256-Val NG2 mutant was incapable of increased motility, while a Thr-2256-Glu mutant was spontaneously motile even in the absence of PMA or PDGF.…”

“…This implies that the site of cytoplasmic phosphorylation of NG2 can determine its ability to interact with cytoplasmic scaffolding proteins that anchor the proteoglycan (and its integrin binding partner) within specific membrane microdomains. MUPP1, 21 GRIP1, 22 syntenin-1 23 and ezrin 24 are possible candidates for such anchoring functions. In this respect, syntenin-1 has been implicated in NG2-dependent oligodendrocyte progenitor migration.…”

Roles of NG2 glial cells in diseases of the central nervous system

“…Moreover, phosphorylated NG2 at Thr2256 is co-localized with α3β1 integrin in broad lamellipodia at the leading edges of motile cells. NG2 phosphorylation at Thr2256 is responsible for relocation of the NG2/integrin complex to lamellipodia, accompanied by increased cell motility [69,70] .…”