Research report
Protein serine/threonine phosphatase 1 and 2A associate with and dephosphorylate neurofilaments

https://doi.org/10.1016/S0169-328X(97)00117-4Get rights and content

Abstract

The phosphorylation state of neurofilaments plays an important role in the control of cytoskeletal integrity, axonal transport, and axon diameter. Immunocytochemical analyses of spinal cord revealed axonal localization of all protein phosphatase subunits. To determine whether protein phosphatases associate with axonal neurofilaments, neurofilament proteins were isolated from bovine spinal cord white matter by gel filtration. ≈15% of the total phosphorylase a phosphatase activity was present in the neurofilament fraction. The catalytic subunits of PP1 and PP2A, as well as the A and Bα regulatory subunits of PP2A, were detected in the neurofilament fraction by immunoblotting, whereas PP2B and PP2C were found exclusively in the low molecular weight soluble fractions. PP1 and PP2A subunits could be partially dissociated from neurofilaments by high salt but not by phosphatase inhibitors, indicating that the interaction does not involve the catalytic site. In both neurofilament and soluble fractions, 75% of the phosphatase activity towards exogenous phosphorylase a could be attributed to PP2A, and the remainder to PP1 as shown with specific inhibitors. Neurofilament proteins were phosphorylated in vitro by associated protein kinases which appeared to include protein kinase A, calcium/calmodulin-dependent protein kinase, and heparin-sensitive and -insensitive cofactor-independent kinases. Dephosphorylation of phosphorylated neurofilament subunits was mainly (60%) catalyzed by associated PP2A, with PP1 contributing minor activity (10–20%). These studies suggest that neurofilament-associated PP1 and PP2A play an important role in the regulation of neurofilament phosphorylation.

Introduction

A primary regulatory mechanism of many cellular processes is the reversible phosphorylation of proteins on serine/threonine residues catalyzed by a complex network of protein serine/threonine kinases and phosphatases. In contrast to the protein kinases in the CNS that have been extensively investigated, less is known about the structure, function, and regulation of protein phosphatases. Recent data, however, has revealed that the phosphatase family of enzymes play an integral role in the control of neuronal function 54, 58, 40.

The protein serine/threonine phosphatases represent a multigene family highly conserved in evolution 7, 39, 12, 50, 70. Based on sequence homologies and biochemical properties, the known phosphatases can be divided into four interrelated families. Three of the families, protein serine/threonine phosphatase types 1, 2A, and 2B (PP1, PP2A, and PP2B, respectively), have significant primary amino-acid sequence homology. In contrast, the phosphatase type 2C (PP2C) is more divergent.

Most protein phosphatase catalytic subunits are associated with regulatory subunits [7]. For example, PP2A is a multimeric enzyme composed of a catalytic subunit (C) and two regulatory subunits (A and the variable or B subunit). The physiological form of PP2A is thought to be a heterotrimer composed of the A and C subunits complexed with a B subunit from one of three families (ABC, AB′C, and AB″C) [70]. Several isoforms also exist for each B subunit family. The physiological significance of this B subunit heterogeneity is not known; however, recent data suggest that these subunits regulate substrate specificity [27], inhibitor sensitivity 27, 69, and targeting of the enzyme to its cellular substrates [57]. The PP1 catalytic subunit also is associated with one of multiple regulatory subunits; some of these regulatory subunits have been shown to direct the catalytic subunit to specific subcellular organelles 6, 7, 25, 1.

Neurofilaments, the principal component of the axonal cytoskeleton, are composed of three subunits in mammals, refered to as low (NF-L), middle (NF-M) and high (NF-H) molecular weight neurofilament proteins 44, 33, 30. Neurofilament proteins are structurally related to one another and to other members of the intermediate filament protein family in that they contain an N-terminal head domain, an α-helical central domain, and a C-terminal tail domain. The size difference between NF-L, NF-M, and NF-H is due to an increasingly large tail domain, which is extensively phosphorylated in NF-M and NF-H in vivo 26, 4. Tail domain phosphorylation is thought to be catalyzed by second messenger-independent kinases, including cdk5 32, 63. The N-terminal head domain of the neurofilament subunits contains phosphorylation sites for protein kinase A, protein kinase C, and CaM kinase II, sites shown to be phosphorylated in NF-L and NF-M in vivo 52, 53.

The precise role of neurofilament phosphorylation is still not completely understood. In vitro assembly/disassembly studies with NF-L 17, 21, 41and in vivo pulse labeling experiments 42, 51, 52, 53have suggested that phosphorylation of the N-terminal head domain by second messenger-dependent kinases prevents premature polymerization of neurofilament subunits in the cell body. The phosphorylation state of the C-terminal tail domain, which forms sidearms that protrude from the neurofilament backbone [20], likely regulates the interaction of neurofilaments with other cytoskeletal elements, such as microtubules [38], and the rate of axonal transport [24]. In addition, tail domain phosphorylation may control inter-filament distance and, indirectly, axon diameter through electrostatic repulsion between the sidearms of adjacent neurofilaments 5, 43.

While neurofilament kinases have been studied extensively, relatively little is known about the protein phosphatases in this system. Two recent reports implicated PP2A as the major phosphatase which dephosphorylates neurofilament proteins. Veeranna et al. showed that NF-H phosphorylated in vitro by cdk5 was efficiently dephosphorylated by skeletal muscle PP2A and by a partially purified PP2A-like activity from spinal cord [68]. Saito et al. reported that a form of PP2A associates with neurofilaments and dephosphorylates NF-L phosphorylated by exogenous protein kinase A [48].

In the present report, we examine the neurofilament-associated protein phosphatases that mediate dephosphorylation of neurofilament proteins phosphorylated by endogenous protein kinases. The data indicate that a heterotrimeric complex of PP2A, consisting of the catalytic subunit (C) and both A and Bα regulatory subunits, is the predominant phosphatase that associates with and dephosphorylates all three neurofilament subunits in vitro. PP1 also is associated with neurofilaments and plays a minor role in their dephosphorylation. This work was previously presented in abstract form [61].

Section snippets

Materials

Phosphorylase b, phosphorylase kinase, dephosphorylated casein, and hexokinase F-300 were obtained from Sigma (St. Louis, MO). Sepharose CL-4B was obtained from Pharmacia LKB Biotech (Uppsala, Sweden). The catalytic and regulatory (RIIα) subunits of protein kinase A were generous gifts from Dr. Jackie Corbin (Vanderbilt University). Inhibitor-1 was kindly provided by Dr. Shirish Shenolikar (Duke University) and thiophosphorylated as described [60]. Calyculin A, microcystin-LR, and okadaic acid

Localization of protein phosphatase subunits in the rat spinal cord

Spinal cord is the classical source of neurofilaments for biochemical studies because it is enriched in axons. Until recently, little was known regarding the distribution of protein phosphatases in the spinal cord and their possible co-localization with neurofilaments. Immunocytochemical analysis of phosphatase expression in the rat spinal cord revealed that the protein phosphatase 2A catalytic (PP2A/C) and A and Bα regulatory (PP2A/A and PP2A/Bα, respectively) subunits exhibit similar

Discussion

The phosphorylation state of neurofilaments and other cytoskeletal proteins are tightly regulated, both temporally and spatially [44]. Indeed, an imbalance of protein kinase and/or phosphatase activities may lead to hyperphosphorylation of tau in neurofibrillary tangles and neurofilament proteins in Lewy bodies, lesions associated with Alzheimer's disease and Lewy body dementia, respectively 45, 66.

In contrast to PP2B, whose expression is highly localized to an axon tract in the dorsolateral

Acknowledgements

This work was supported by National Institutes of Health Grants GM51366 (to B.E.W.), NS13031 (to F.F.E.), and GM47973 (to R.J.C.). B.E.W. is the recipient of a Faculty Development Award from the Pharmaceutical Research and Manufacturers of America Foundation. R.J.C. is an Established Investigator of the AHA. We thank Dr. Robley Williams for helpful discussions.

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