Phosphorylation of GAP-43 T172 is a molecular marker of growing axons in a wide range of mammals including primates

AbstractGAP-43 is a vertebrate neuron-specific protein and that is strongly related to axon growth and regeneration; thus, this protein has been utilized as a classical molecular marker of these events and growth cones. Although GAP-43 was biochemically characterized more than a quarter century ago, how this protein is related to these events is still not clear. Recently, we identified many phosphorylation sites in the growth cone membrane proteins of rodent brains. Two phosphorylation sites of GAP-43, S96 and T172, were found within the top 10 hit sites among all proteins. S96 has already been characterized (Kawasaki et al., 2018), and here, phosphorylation of T172 was characterized. In vitro (cultured neurons) and in vivo, an antibody specific to phosphorylated T172 (pT172 antibody) specifically recognized cultured growth cones and growing axons in developing mouse neurons, respectively. Immunoblotting showed that pT172 antigens were more rapidly downregulated throughout development than those of pS96 antibody. From the primary structure, this phosphorylation site was predicted to be conserved in a wide range of animals including primates. In the developing marmoset brainstem and in differentiated neurons derived from human induced pluripotent stem cells, immunoreactivity with pT172 antibody revealed patterns similar to those in mice. pT172 antibody also labeled regenerating axons following sciatic nerve injury. Taken together, the T172 residue is widely conserved in a wide range of mammals including primates, and pT172 is a new candidate molecular marker for growing axons.

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A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

Molecular and Cellular Biology ◽

10.1128/mcb.15.10.5214 ◽

1995 ◽

Vol 15 (10) ◽

pp. 5214-5225 ◽

Cited By ~ 137

Author(s):

A D Catling ◽

H J Schaeffer ◽

C W Reuter ◽

G R Reddy ◽

M J Weber

Keyword(s):

Protein Interactions ◽

Critical Role ◽

Phosphorylation Site ◽

Specific Protein ◽

Protein Protein Interactions ◽

Serum Stimulation ◽

And Function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

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The Arabidopsis Rho of Plants GTPase ROP1 Is a Potential Calcium-Dependent Protein Kinase (CDPK) Substrate

Plants ◽

10.3390/plants10102053 ◽

2021 ◽

Vol 10 (10) ◽

pp. 2053

Author(s):

Dalma Ménesi ◽

Éva Klement ◽

Györgyi Ferenc ◽

Attila Fehér

Keyword(s):

Protein Kinases ◽

Phosphorylation Site ◽

Interaction Strength ◽

Yeast Cells ◽

Phosphorylation Sites ◽

Mutant Proteins ◽

In Vivo Test ◽

Calcium Dependent

Plant Rho-type GTPases (ROPs) are versatile molecular switches involved in a number of signal transduction pathways. Although it is well known that they are indirectly linked to protein kinases, our knowledge about their direct functional interaction with upstream or downstream protein kinases is scarce. It is reasonable to suppose that similarly to their animal counterparts, ROPs might also be regulated by phosphorylation. There is only, however, very limited experimental evidence to support this view. Here, we present the analysis of two potential phosphorylation sites of AtROP1 and two types of potential ROP-kinases. The S74 site of AtROP1 has been previously shown to potentially regulate AtROP1 activation dependent on its phosphorylation state. However, the kinase phosphorylating this evolutionarily conserved site could not be identified: we show here that despite of the appropriate phosphorylation site consensus sequences around S74 neither the selected AGC nor CPK kinases phosphorylate S74 of AtROP1 in vitro. However, we identified several phosphorylation sites other than S74 for the CPK17 and 34 kinases in AtROP1. One of these sites, S97, was tested for biological relevance. Although the mutation of S97 to alanine (which cannot be phosphorylated) or glutamic acid (which mimics phosphorylation) somewhat altered the protein interaction strength of AtROP1 in yeast cells, the mutant proteins did not modify pollen tube growth in an in vivo test.

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Engineering and Characterisation of Bacterial Phosphopantetheinyl Transferases and their Peptide Substrates

10.26686/wgtn.17060873 ◽

2021 ◽

Author(s):

◽

Jack Alexander Sissons

Keyword(s):

Single Gene ◽

Specific Protein ◽

Rapid Identification ◽

Phosphopantetheinyl Transferase ◽

Post Translational Modification ◽

Diverse Range ◽

Peptide Motifs ◽

Wide Range

<p>Throughout all domains of life, phosphopantetheinyl transferase (PPTase) enzymes catalyse a post-translational modification that is important in both primary and secondary metabolism; the transfer of a phosphopantetheine (PPant) group derived from Coenzyme A to specific protein domains within large, multi-modular biosynthetic enzymes, thereby activating each module for biosynthesis. The short peptide motif of the protein to which this group is attached is known as a ‘tag’, and can be fused to other proteins, making them also substrates for post-translational modification by a PPTase. Additionally, it has been demonstrated that PPTases can utilise a diverse range of CoA analogues, such as biotin-linked or click-chemistry capable CoA derivatives, as substrates for tag attachment. Together, these characteristics make post-translational modification by PPTases an attractive system for many different biotechnological applications. Perhaps the most significant application is in vivo and in vitro site-specific labelling of proteins, for which current technologies are hindered by cumbersome fusion protein requirements, toxicity of the process, or limited reporter groups that can be attached. Confoundingly, most PPTases exhibit a high degree of substrate promiscuity which limits the number of PPTase-tag pairs that can be used simultaneously, and therefore the number of protein targets that can be simultaneously labelled. To address this, directed evolution at a single gene level was used in an attempt to generate multiple PPTase variants that have non-overlapping tag specificity which have applications in orthogonal labelling. Furthermore, assays for the rapid identification, characterisation and evolution of short, novel peptide motifs that are recognised by PPTases has further diversified the labelling toolkit. These developments have enhanced the utility of the PPTase system and potentially have a wide range of applications in a number of fields.</p>

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Phosphorylation-site mutagenesis of the growth-associated protein GAP-43 modulates its effects on cell spreading and morphology.

The Journal of Cell Biology ◽

10.1083/jcb.120.2.503 ◽

1993 ◽

Vol 120 (2) ◽

pp. 503-512 ◽

Cited By ~ 71

Author(s):

F Widmer ◽

P Caroni

Keyword(s):

Phosphorylation Site ◽

Growth Cones ◽

Cell Cortex ◽

Membrane Association ◽

Major Protein ◽

Cell Periphery ◽

Cortical Actin ◽

Cortical Cytoskeleton

The 43-kD growth-associated protein (GAP-43) is a major protein kinase C (PKC) substrate of axonal growth cones, developing nerve terminals, regenerating axons, and adult central nervous system areas associated with plasticity. It is a cytosolic protein associated with the cortical cytoskeleton and the plasmalemma. Membrane association of GAP-43 is mediated by palmitoylation at Cys3Cys4. In vitro and in vivo, phosphorylation by PKC exclusively involves Ser41 of mammalian GAP-43 (corresponding to Ser42 in the chick protein). To identify aspects of GAP-43 function, we analyzed the actions of wild-type, membrane-association, and phosphorylation-site mutants of GAP-43 in nonneuronal cell lines. The GAP-43 constructs were introduced in L6 and COS-7 cells by transient transfection. Like the endogenous protein in neurons and their growth cones, GAP-43 in nonneuronal cells associated with the cell periphery. GAP-43 accumulated in the pseudopods of spreading cells and appeared to interact with cortical actin-containing filaments. Spreading L6 cells expressing high levels of recombinant protein displayed a characteristic F-actin labeling pattern consisting of prominent radial arrays of peripheral actin filaments. GAP-43 had dramatic effects on local surface morphology. Characteristic features of GAP-43-expressing cells were irregular cell outlines with prominent and numerous filopodia. The effects of GAP-43 on cell morphology required association with the cell membrane, since GAP-43(Ala3Ala4), a mutant that failed to associate with the cell cortex, had no morphogenetic activity. Two GAP-43 phosphorylation mutants (Ser42 to Ala42 preventing and Ser42 to Asp42 mimicking phosphorylation by PKC) modulated the effects of GAP-43 in opposite ways. Cells expressing GAP-43(Asp42) spread extensively and displayed large and irregular membranous extensions with little filopodia, whereas GAP-43(Ala42) produced small, poorly spreading cells with numerous short filopodia. Therefore, GAP-43 influences cell surface behavior and phosphorylation modulates its activity. The presence of GAP-43 in growing axons and developing nerve termini may affect the behavior of their actin-containing cortical cytoskeleton in a regulatable manner.

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Microfluidic neural axon diode

TECHNOLOGY ◽

10.1142/s2339547816500102 ◽

2016 ◽

Vol 04 (04) ◽

pp. 240-248 ◽

Cited By ~ 8

Author(s):

Sangcheol Na ◽

Myeongwoo Kang ◽

Seokyoung Bang ◽

Daehun Park ◽

Jinhyun Kim ◽

...

Keyword(s):

Neural Circuit ◽

Axon Growth ◽

In Vivo Models ◽

Starting Point ◽

Wide Range ◽

Reproducible Method ◽

Physical And Chemical ◽

Biology Research

Neural circuits, groups of neurons connected in directional manner, play a central role in information processing. Advances in neuronal biology research is limited by a lack of appropriate in vitro methods to construct and probe neuronal networks. Here, we describe a microfluidic culture platform that directs the growth of axons using “neural diode” structures to control neural connectivity. This platform is compatible with live cell imaging and can be used to (i) form pre-synaptic and postsynaptic neurons by directional axon growth and (ii) localize physical and chemical treatment to pre- or postsynaptic neuron groups (i.e. virus infection and etc.). The “neural diode” design consist of a microchannel that split into two branches: one is directed straight toward while the other returns back toward the starting point in a closed loop to send the axons back to the origin. We optimized the “neural diode” pattern dimension and design to achieve close to 70% directionality with a single unit of the “diode”. When repeated 3 times, near perfect (98–100% at wide range of cell concentrations) directionality can be achieved. The living neural circuit was characterized using Ca imaging and confirmed their function. The platform also serves as a straightforward, reproducible method to recapitulate a variety of neural circuit in vitro that were previously observable only in brain slice or in vivo models. The microfluidic neural diode may lead to better models for understanding the neural circuit and neurodegenerative diseases.

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Rad53 Checkpoint Kinase Phosphorylation Site Preference Identified in the Swi6 Protein of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.23.10.3405-3416.2003 ◽

2003 ◽

Vol 23 (10) ◽

pp. 3405-3416 ◽

Cited By ~ 32

Author(s):

Julia M. Sidorova ◽

Linda L. Breeden

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Phosphorylation Site ◽

Site Preference ◽

Phosphorylation Sites ◽

Checkpoint Kinase ◽

Checkpoint Signaling ◽

And Function

ABSTRACT Rad53 of Saccharomyces cerevisiae is a checkpoint kinase whose structure and function are conserved among eukaryotes. When a cell detects damaged DNA, Rad53 activity is dramatically increased, which ultimately leads to changes in DNA replication, repair, and cell division. Despite its central role in checkpoint signaling, little is known about Rad53 substrates or substrate specificity. A number of proteins are implicated as Rad53 substrates; however, the evidence remains indirect. Previously, we have provided evidence that Swi6, a subunit of the Swi4/Swi6 late-G1-specific transcriptional activator, is a substrate of Rad53 in the G1/S DNA damage checkpoint. In the present study we identify Rad53 phosphorylation sites in Swi6 in vitro and demonstrate that at least one of them is targeted by Rad53 in vivo. Mutations in these phosphorylation sites in Swi6 shorten but do not eliminate the Rad53-dependent delay of the G1-to-S transition after DNA damage. We derive a consensus for Rad53 site preference at positions −2 and +2 (−2/+2) and identify its potential substrates in the yeast proteome. Finally, we present evidence that one of these candidates, the cohesin complex subunit Scc1 undergoes DNA damage-dependent phosphorylation, which is in part dependent on Rad53.

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Stem cells: an origin and marks

Vestnik of Russian military medical Academy ◽

10.17816/brmma50075 ◽

2020 ◽

Vol 22 (2) ◽

pp. 211-216

Author(s):

A. V. Moskalev ◽

B. Y. Gumilevskiy ◽

A. V. Apchel ◽

V. N. Cygan

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Molecular Marker ◽

Population Level ◽

Cell Types ◽

Stem Cell Marker ◽

Wide Range ◽

Almost All

The basic physiological functions of stem cells are given: the ability to reproduce and generate offspring, which are manifested at the level of the population, and not of a single cell. The manifestation of these functions depends on the quantitative and qualitative composition of the microenvironment. Stem cells consist of two fundamentally different types: pluripotent, which exist only in vitro (in vitro) and tissue, existing in the postpartum body (in vivo). Stem cells can be replaced without limitation in vitro and lead to the appearance of a wide range of cell types. Tissue stem cells under normal conditions do not generate cells characteristic of other types of tissue. Stem cells include cells capable of expressing the gene products characteristic of them. However, there is no universal marker to differentiate stem cells from non-stem cells. A key marker of pluripotency is the transcription factor - a pituitary-specific transcription factor is positive. A component that can be found in almost all types of stem cells is the telomerase complex. Another stem cell marker is called CD34 glycoprotein. The functional activity of stem cells is associated with a molecular marker referred to as leucine-rich repeat containing G-protein bound to receptor 5. However, other types of cells do not express this marker. The physiological capabilities of stem cells depend both on the cells themselves and on their environment. The most reliable way to identify stem cells is to determine their phenotype in vivo. This suggests that stem cells do not carry a universal molecular marker. Most likely, they have significant differences from transplanted cells, and these differences cannot always be detected in individual cells, but only at the population level.

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Engineering and Characterisation of Bacterial Phosphopantetheinyl Transferases and their Peptide Substrates

10.26686/wgtn.17060873.v1 ◽

2021 ◽

Author(s):

◽

Jack Alexander Sissons

Keyword(s):

Single Gene ◽

Specific Protein ◽

Rapid Identification ◽

Phosphopantetheinyl Transferase ◽

Post Translational Modification ◽

Diverse Range ◽

Peptide Motifs ◽

Wide Range

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The relative roles of specific N- and C-terminal phosphorylation sites in the disassembly of intermediate filament in mitotic BHK-21 cells

Journal of Cell Science ◽

10.1242/jcs.109.4.817 ◽

1996 ◽

Vol 109 (4) ◽

pp. 817-826 ◽

Cited By ~ 1

Author(s):

Y.H. Chou ◽

P. Opal ◽

R.A. Quinlan ◽

R.D. Goldman

Keyword(s):

Protein Kinase ◽

Phosphorylation Site ◽

Supramolecular Organization ◽

Phosphorylation Sites ◽

Bhk Cells ◽

Terminal Domain ◽

Kinase Phosphorylation ◽

Mitotic Cells

Previously we identified p34cdc2 as one of two protein kinases mediating the hyperphosphorylation and disassembly of vimentin in mitotic BHK-21 cells. In this paper, we identify the second kinase as a 37 kDa protein. This p37 protein kinase phosphorylates vimentin on two adjacent residues (thr-457 and ser-458) which are located in the C-terminal non-alpha-helical domain. Contrary to the p34cdc2 mediated N-terminal phosphorylation (at ser-55) which can disassemble vimentin intermediate filaments (IF) in vitro, p37 protein kinase phosphorylates vimentin-IF without obviously affecting its structure in vitro. We have further examined the in vivo role(s) of vimentin phosphorylation in the disassembly of the IF network in mitotic BHK cells by transient transfection assays. In untransfected BHK cells, the interphase vimentin IF networks are disassembled into non-filamentous aggregates when cells enter mitosis. Transfection of cells with vimentin cDNA lacking the p34cdc2 phosphorylation site (ser55:ala) effectively prevents mitotic cells from disassembling their IF. In contrast, apparently normal disassembly takes place in cells transfected with cDNA containing mutated p37 kinase phosphorylation sites (thr457:ala/ser458:ala). Transfection of cells with vimentin cDNAs lacking both the N- and C-terminal phosphorylation sites yields a phenotype indistinguishable from that obtained with the single N-terminal mutant. Taken together, our results demonstrate that the site-specific phosphorylation of the N-terminal domain, but not the C-terminal domain of vimentin plays an important role in determining the state of IF polymerization and supramolecular organization in mitotic cells.

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Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5

Biochemical Journal ◽

10.1042/bj20040324 ◽

2004 ◽

Vol 381 (2) ◽

pp. 471-481 ◽

Cited By ~ 28

Author(s):

Mark E. GRAHAM ◽

Patricia RUMA-HAYNES ◽

Amanda G. CAPES-DAVIS ◽

Joanne M. DUNN ◽

Timothy C. TAN ◽

...

Keyword(s):

Phosphorylation Site ◽

Site Directed Mutagenesis ◽

Phosphorylation Sites ◽

Single Mutation ◽

Cyclin Dependent Kinase ◽

Major Site ◽

Multisite Phosphorylation ◽

Cyclin Dependent Kinase 5

Doublecortin (DCX) is a 40 kDa microtubule-associated protein required for normal neural migration and cortical layering during development. Mutations in the human DCX gene cause a disruption of cortical neuronal migration. Defects in cdk5 (cyclin-dependent kinase 5) also cause defects in neural migration and cortical layering. DCX is a substrate for cdk5 in vitro and in vivo and the major site of in vitro phosphorylation is Ser-297. We used a highly developed MS strategy to identify the cdk5 phosphorylation sites and determine the major and minor sites. Several phosphopeptides were identified from a tryptic digest of 32P-labelled, cdk5-phosphorylated DCX using a combination of off-line HPLC and matrix-assisted laser-desorption ionization-MS with alkaline phosphatase treatment. Tandem MS/MS enabled the identification of seven phosphorylation sites for cdk5. Monitoring of 32P label indicated that there was one major site, Ser-28, at the N-terminus, and a major site, Ser-339, in the serine/proline-rich domain at the C-terminus. Five other sites, Ser-287, Thr-289, Ser-297, Thr-326 and Ser-332, were also found in the tail. Site-directed mutagenesis largely supported these findings. Single mutation of Ser-28 reduced but did not abolish phosphorylation. Double, rather than single, mutation for Ser-332 and Ser-339 was required to reduce overall phosphorylation, suggesting an interaction between these sites. Truncations of the tail produced a significant reduction in cdk5 phosphorylation of DCX. These results do not support Ser-297 as the major cdk5 phosphorylation site in DCX, but indicate that DCX is subject to complex multisite phosphorylation. This illustrates the importance of a well-developed MS strategy to identify phosphorylation sites.

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