Research reportProtein serine/threonine phosphatase 1 and 2A associate with and dephosphorylate neurofilaments
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.
References (70)
- et al.
Properties of several protein kinases that copurify with rat spinal cord neurofilaments
Biochim. Biophys. Acta
(1989) - et al.
The structure, biochemical properties, and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state
J. Biol. Chem.
(1985) - et al.
Identification of a third form of protein phosphatase 1 in rabbit skeletal muscle that is associated with myosin
Biochim. Biophys. Acta
(1988) Classification of protein-serine/threonine phosphatases: identification and quantitation in cell extracts
Methods Enzymol.
(1991)- et al.
Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle
Methods Enzymol.
(1988) - et al.
Okadaic acid: a new probe for the study of cellular regulation
Trends Biochem. Sci.
(1990) - et al.
Serine/threonine protein phosphatases in the control of cell function
Adv. Enzyme Regul.
(1994) - et al.
The catalytic subunit of protein phosphatase 2A is carboxyl-methylated in vivo
J. Biol. Chem.
(1994) - et al.
Involvement of protein kinase C in the regulation of assembly-disassembly of neurofilaments in vitro
Biochem. Biophys. Res. Commun.
(1990) - et al.
Structure of the peripheral domains of neurofilaments revealed by low angle rotary shadowing
J. Mol. Biol.
(1988)
The distribution of phosphorylation sites among identified proteolytic fragments of mammalian neurofilaments
J. Biol. Chem.
Subunit interactions control protein phosphatase 2A. Effects of limited proteolysis, N-ethylmaleimide, and heparin on the interaction of the B subunit
J. Biol. Chem.
Antibodies against a synthetic peptide as a probe for the kinase activity of the EGF receptor and v-erb protein
Cell
Neurofilament function and dysfunction: involvement in axonal growth and neuronal disease
Curr. Opin. Cell Biol.
Neuronal cdc2-like kinase
Trends Biochem. Sci.
Molecular biology of neuronal intermediate filaments
Curr. Opin. Cell Biol.
Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants
FEBS Lett.
Phosphorylation of tau protein by purified p34cdc28 and a related protein kinase from neurofilaments
J. Biol. Chem.
Protein phosphatase-2C from rabbit skeletal muscle and liver: an Mg2+-dependent enzyme
Methods Enzymol.
Interaction of the tail domain of high molecular weight subunits of neurofilaments with the COOH-terminal region of tubulin and its regulation by tau protein kinase II
J. Biol. Chem.
The role of protein phosphatases in synaptic transmission, plasticity and neuronal development
Curr. Opin. Neurobiol.
Effect of phosphorylation on 68 kDa neurofilament subunit protein assembly by the cyclic AMP dependent protein kinase in vitro
Biochem. Biophys. Res. Commun.
Differential turnover of phosphate groups on neurofilament subunits in mammalian neurons in vivo
J. Biol. Chem.
Neurofilament phosphorylation: a new look at regulation and function
Trends Neurosci.
Okadaic acid induces the rapid and reversible disruption of the neurofilament network in rat dorsal root ganglion neurons
Biochem. Biophys. Res. Commun.
Increased phosphorylation of the amino-terminal domain of the low molecular weight neurofilament subunit in okadaic acid-treated neurons
J. Biol. Chem.
In vivo phosphorylation of distinct domains of the 70-kilodalton neurofilament subunit involves different protein kinases [Published erratum appeared in J. Biol. Chem., 264 (7), 4264, 5 March 1989.]
J. Biol. Chem.
Phosphorylation of the amino-terminal head domain of the middle molecular mass 145-kDa subunit of neurofilaments. Evidence for regulation by second messenger-dependent protein kinases
J. Biol. Chem.
Identification of Ser-55 as a major protein kinase A phosphorylation site on the 70-kDa subunit of neurofilaments. Early turnover during axonal transport
J. Biol. Chem.
Functional determinants in the autoinhibitory domain of calcium/calmodulin-dependent protein kinase II:. Role of His282 and multiple basic residues
J. Biol. Chem.
Serine/threonine phosphatases in the nervous system
Curr. Opin. Neurobiol.
Protein phosphatase-2B from rabbit skeletal muscle: a Ca2+-dependent, calmodulin-stimulated enzyme
Methods Enzymol.
Phosphorylation of the high molecular weight neurofilament protein (NF-H) by cdk5 and p35
J. Biol. Chem.
The structure, role, and regulation of type 1 protein phosphatases
Crit. Rev. Biochem. Mol. Biol.
Axonal tubulin and axonal microtubules: biochemical evidence for cold stability
J. Cell Biol.
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