Identification of Ser‐Pro and Thr‐Pro Phosphorylation Sites in Chicken Neurofilament‐M Tail Domain

Bennett, Gudrun S.; Quintana, Ricardo

doi:10.1046/j.1471-4159.1997.68020534.x

Cited by 18 publications

(17 citation statements)

References 25 publications

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“…(ii) 2D-gel electrophoresis of radioactive samples can rapidly pinpoint by autoradiography the phosphorylated proteins and the changes in the extent of phosphorylation. The protein spots of interest can be excised, digested, and analyzed by MS (Greenwood et al, 1994;Watts et al, 1994;Bennett & Quintana, 1997;Kang et al, 1997;Neubauer & Mann, 1999). Figure 4 shows the phosphopeptide analysis and phosphorylation site determination of an in-gel digested Op18 protein after in vitro phosphorylation (Neubauer & Mann, 1999).…”

Section: Emergence Of Phosphoproteomicsmentioning

confidence: 99%

Phosphoproteomics by mass spectrometry and classical protein chemistry approaches

Salih

2004

Mass Spectrometry Reviews

119

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The general fields of biological sciences have seen phenomenal transformations in the past two decades at the level of data acquisition, understanding biological processes, and technological developments. Those advances have been made partly because of the advent of molecular biology techniques (which led to genomics) coupled to the advances made in mass spectrometry (MS) to provide the current capabilities and developments in proteomics. However, our current knowledge that approximately 30,000 human genes may code for up to 1 million or more proteins disengage the interface between the genome sequence database algorithms and MS to generate a major interest in independent de novo MS/MS sequence determination. Significant progress has been made in this area through procedures to covalently modify peptide N- and C-terminal amino-acids by sulfonation and guanidination to permit rapid de novo sequence determination by MS/MS analysis. A number of strategies that have been developed to perform qualitative and quantitative proteomics range from 2D-gel electrophoresis, affinity tag reagents, and stable-isotope labeling. Those procedures, combined with MS/MS peptide sequence analysis at the subpicomole level, permit the rapid and effective identification and quantification of a large number of proteins within a given biological sample. The identification of proteins per se, however, is not always sufficient to interpret biological function because many of the naturally occurring proteins are post-translationally modified. One such modification is protein phosphorylation, which regulates a large array of cellular biochemical pathways of the biological system. Traditionally, the study of phosphoprotein structure-function relationships involved classical protein chemistry approaches that required protein purification, peptide mapping, and the identification of the phosphorylated peptide regions and sites by N-terminal sequence analysis. Recent advances made in mass spectrometry have clearly revolutionized the studies of phosphoprotein biochemistry, and include the development of specific strategies to preferentially enrich phosphoproteins by covalent-modifications that incorporate affinity tags that use the physicochemical properties of phosphoaminoacids. The phosphoserine/phosphothreonine-containing proteins/peptides are derivatized under base-catalyzed conditions by thiol agents; mono- and di-thiol reagents both have been used in such studies. The thiol agent may have: (i) an affinity tag for protein enrichment; (ii) stable-isotopic variants for relative quantitation; or (iii) a combination of the moieties in (i) and (ii). These strategies and techniques, together with others, are reviewed, including their practical application to the study of phosphoprotein biochemistry and structure-function. The consensus of how classical protein chemistry and current MS technology overlap into special case of proteomics, namely "phosphoproteomics," will be discussed.

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Section: Emergence Of Phosphoproteomicsmentioning

confidence: 99%

Phosphoproteomics by mass spectrometry and classical protein chemistry approaches

Salih

2004

Mass Spectrometry Reviews

119

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“…Second messenger-independent kinases such as casein kinase I and II (CKI and CKII) are found in NF preparations from mammalian brain tissue. CKI, a constitutively active kinase, is the principal kinase associated with NFs in preparations derived from squid axoplasm, bovine and chicken spinal cord, and in vitro studies show that CKI phosphorylates serine/threonine residues in the glutamic-acid-rich C-terminal domain of NFs [25][26][27][28][29][30][31]. Another kinase enriched in squid NF preparations is calcium-calmodulin kinase II (CamKII), which has also been shown to phosphorylate NF proteins, among other substrates in the postsynaptic density protein complex [32].…”

Section: Phosphorylation Of Nfsmentioning

confidence: 99%

Cyclin-Dependent Kinase 5 in Neurofilament Function and Regulation

Kesavapany

Pant³

2003

Neurosignals

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Neurofilaments are neuron-specific intermediate filaments. They are classed into three groups according to their molecular masses: neurofilament heavy, middle and light chains (NF-H, NF-M and NF-L). Neurofilaments assemble and form through the association of their central α-helical coiled-coil rod domains. NF-H and NF-M are distinct from NF-L as they contain a carboxyl-terminal tail domain, which appears to form connections with adjacent structures and other neurofilaments. Together with other axonal components such as microtubules, they form the dynamic axonal cytoskeleton. They maintain and regulate neuronal cytoskeletal plasticity through the regulation of neurite outgrowth, axonal caliber and axonal transport. Neurofilaments contain KSP repeats that are consensus motifs for the proline-directed kinases and are extensively phosphorylated in vivo, and their functions are thought to be regulated through their phosphorylation. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed kinase, whose activity is restricted to the neuron through the neuronal-specific distribution of its activators p35 and p39. Cdk5 is the only kinase that affects the electrophoretic mobility of human NF-H and is thought to be the major neurofilament kinase. Cdk5 is involved in crosstalk with other signal transduction pathways such as the mitogen-activated protein kinase and myelin-associated glycoprotein pathways to influence the phosphorylation of neurofilaments and other cytoskeletal proteins. Both the hyperactivation of Cdk5 activity and subsequent hyperphosphorylation of neurofilaments and the microtubule-associated protein tau have been implicated in the pathogenesis of neurodegenerative disorders such as Alzheimer’s disease and amyotrophic lateral sclerosis. Here we review the functions of neurofilaments and the significance of Cdk5 phosphorylation of neurofilaments.

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“…1A). At least 21, and probably all 22, are phosphorylated in vivo (Bennett and Quintana, 1997). Five additional serines in the acidic amino end of the tail are also phosphorylated in vivo (Ser 464 , Ser 471 , Ser 502 , Ser 528 , and Ser 536 ; Fig.…”

Section: Resultsmentioning

confidence: 99%

“… A : Diagram of chicken NFM [modified from Bennett and Quintana (1997)] and the CM : 381‐558 fusion protein. Several distinct phosphorylation motifs are contained in the tail domain of chicken NF‐M, including five CKI sites, four KSP repeats, 17 KXX(S/T)P repeats, and one SP site.…”

mentioning

confidence: 99%

Relationship Between Casein Kinase I Isoforms and a Neurofilament‐Associated Kinase

Green²,

Bennett³

1999

Journal of Neurochemistry

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Purified neurofilaments (NFs) contain an associated kinase (NFAK) activity that phosphorylates selectively a subset of sites in the tail of NF-M and has properties consistent with casein kinase I (CKI). Because CKI consists of a family of as many as seven genes (␣, ␤, ␥1-3, ␦, and ⑀), we investigated the extent to which different CKI isoforms contribute to NFAK activity. Using an NF-M-derived substrate, we determined that NFAK activity copurified with casein kinase activity through two purification steps. In an in-gel kinase assay, NFAK activity occurred at 36 -40 kDa, corresponding to the size of CKI␣ isoforms. Chicken neurons express transcripts encoding four alternatively spliced variants of CKI␣ (CKI␣, CKI␣S, CKI␣L, and CKI␣LS) differing in the presence or absence of two inserts, L and S. Using antibodies against different isoforms or with broad CKI specificity, we determined that all four CKI␣ variants, as well as other CKI family members, are present in chicken brain. However, only CKI␣ and CKI␣S could be detected in purified NFAK. Also, immunoprecipitation studies showed that CKI␣ and CKI␣S together account for NFAK activity. These findings raise the possibility that only a subset of CKI isoforms may be able to associate with and/or phosphorylate NFs.

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Identification of Ser‐Pro and Thr‐Pro Phosphorylation Sites in Chicken Neurofilament‐M Tail Domain

Cited by 18 publications

References 25 publications

Phosphoproteomics by mass spectrometry and classical protein chemistry approaches

Phosphoproteomics by mass spectrometry and classical protein chemistry approaches

Cyclin-Dependent Kinase 5 in Neurofilament Function and Regulation

Relationship Between Casein Kinase I Isoforms and a Neurofilament‐Associated Kinase

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