scholarly journals Regulation of Plant Phototropic Growth by NPH3/RPT2-like Substrate Phosphorylation and 14-3-3 Binding

2021 ◽  
Author(s):  
Stuart Sullivan ◽  
Thomas Waksman ◽  
Louise Henderson ◽  
Dimitra Paliogianni ◽  
Melanie Lütkemeyer ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Common Mechanism ◽  
Binding Motif ◽  
Growth Responses ◽  
Directional Growth ◽  
Agc Kinases ◽  
C Terminus

Polarity underlies all plant physiology and directional growth responses such as phototropism. Yet, our understanding of how plant tropic responses are established is far from complete. The plasma-membrane associated BTB-containing protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key determinant of phototropic growth which is regulated by AGC kinases known as the phototropins (phots). However, the mechanism by which phots initiate phototropic signalling via NPH3, and other NPH3/RPT2-like (NRL) members, has remained unresolved. Here we demonstrate that NPH3 is directly phosphorylated by phot1 both in vitro and in vivo. Light-dependent phosphorylation within a conserved consensus sequence (RxS) located at the extreme C-terminus of NPH3 is necessary to promote its functionality for phototropism and petiole positioning in Arabidopsis. Phosphorylation of this region by phot1 also triggers 14-3-3 binding combined with changes in NPH3 phosphorylation and localisation status. Seedlings expressing mutants of NPH3 that are unable to bind or constitutively bind 14-3-3s show compromised functionality that is consistent with a model where signalling outputs arising from a gradient in NPH3 RxS phosphorylation/localisation across the stem are a major contributor to phototropic responsiveness. Our current findings provide further evidence that 14-3-3 proteins are instrumental components regulating auxin-dependent growth and show for the first time that NRL proteins are direct phosphorylation targets for plant AGC kinases. Moreover, the C-terminal phosphorylation site/14-3-3-binding motif of NPH3 is conserved in several members of the NRL family, suggesting a common mechanism of regulation.

scholarly journals Substrate priming enhances phosphorylation by the budding yeast kinases Kin1 and Kin2

2018 ◽  
Vol 293 (47) ◽  
pp. 18353-18364
Author(s):  
Grace R. Jeschke ◽  
Hua Jane Lou ◽  
Keith Weise ◽  
Charlotte I. Hammond ◽  
Mallory Demonch ◽  
...  
Keyword(s):  
Er Stress ◽  
Secretory Pathway ◽  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Growth Defect ◽  
Common Mechanism ◽  
Sequence Motif ◽  
Dependent Manner

Multisite phosphorylation of proteins is a common mechanism for signal integration and amplification in eukaryotic signaling networks. Proteins are commonly phosphorylated at multiple sites in an ordered manner, whereby phosphorylation by one kinase primes the substrate by generating a recognition motif for a second kinase. Here we show that substrate priming promotes phosphorylation by Saccharomyces cerevisiae Kin1 and Kin2, kinases that regulate cell polarity, exocytosis, and the endoplasmic reticulum (ER) stress response. Kin1/Kin2 phosphorylated substrates within the context of a sequence motif distinct from those of their most closely related kinases. In particular, the rate of phosphorylation of a peptide substrate by Kin1/Kin2 increased >30-fold with incorporation of a phosphoserine residue two residues downstream of the phosphorylation site. Recognition of phosphorylated substrates by Kin1/Kin2 was mediated by a patch of basic residues located in the region of the kinase αC helix. We identified a set of candidate Kin1/Kin2 substrates reported to be dually phosphorylated at sites conforming to the Kin1/Kin2 consensus sequence. One of these proteins, the t-SNARE protein Sec9, was confirmed to be a Kin1/Kin2 substrate both in vitro and in vivo. Sec9 phosphorylation by Kin1 in vitro was enhanced by prior phosphorylation at the +2 position. Recognition of primed substrates was not required for the ability of Kin2 to suppress the growth defect of secretory pathway mutants but was necessary for optimal growth under conditions of ER stress. These results suggest that at least some endogenous protein substrates of Kin1/Kin2 are phosphorylated in a priming-dependent manner.

scholarly journals Regulation of plant phototropic growth by NPH3/RPT2-like substrate phosphorylation and 14-3-3 binding

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Stuart Sullivan ◽  
Thomas Waksman ◽  
Dimitra Paliogianni ◽  
Louise Henderson ◽  
Melanie Lütkemeyer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plant Growth ◽  
Consensus Sequence ◽  
Light Environment ◽  
Common Mechanism ◽  
Growth Responses ◽  
Directional Growth ◽  
Agc Kinases ◽  
Substrate Phosphorylation

AbstractPolarity underlies all directional growth responses in plants including growth towards the light (phototropism). The plasma-membrane associated protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key determinant of phototropic growth which is regulated by phototropin (phot) AGC kinases. Here we demonstrate that NPH3 is directly phosphorylated by phot1 within a conserved C-terminal consensus sequence (RxS) that is necessary to promote phototropism and petiole positioning in Arabidopsis. RxS phosphorylation also triggers 14-3-3 binding combined with changes in NPH3 phosphorylation and localisation status. Mutants of NPH3 that are unable to bind or constitutively bind 14-3-3 s show compromised functionality consistent with a model where phototropic curvature is established by signalling outputs arising from a gradient of NPH3 RxS phosphorylation across the stem. Our findings therefore establish that NPH3/RPT2-Like (NRL) proteins are phosphorylation targets for plant AGC kinases. Moreover, RxS phosphorylation is conserved in other members of the NRL family, suggesting a common mechanism of regulating plant growth to the prevailing light environment.

scholarly journals ParB of Pseudomonas aeruginosa: Interactions with Its Partner ParA and Its Target parS and Specific Effects on Bacterial Growth

2004 ◽  
Vol 186 (20) ◽  
pp. 6983-6998 ◽  
Author(s):  
Aneta A. Bartosik ◽  
Krzysztof Lasocki ◽  
Jolanta Mierzejewska ◽  
Christopher M. Thomas ◽  
Grazyna Jagura-Burdzy
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Streptomyces Coelicolor ◽  
Consensus Sequence ◽  
Terminal Part ◽  
Dimerization Domain ◽  
Interaction Domain ◽  
C Terminus ◽  
Cell Components

ABSTRACT The par genes of Pseudomonas aeruginosa have been studied to increase the understanding of their mechanism of action and role in the bacterial cell. Key properties of the ParB protein have been identified and are associated with different parts of the protein. The ParB- ParB interaction domain was mapped in vivo and in vitro to the C-terminal 56 amino acids (aa); 7 aa at the C terminus play an important role. The dimerization domain of P. aeruginosa ParB is interchangeable with the dimerization domain of KorB from plasmid RK2 (IncP1 group). The C-terminal part of ParB is also involved in ParB-ParA interactions. Purified ParB binds specifically to DNA containing a putative parS sequence based on the consensus sequence found in the chromosomes of Bacillus subtilis, Pseudomonas putida, and Streptomyces coelicolor. The overproduction of ParB was shown to inhibit the function of genes placed near parS. This “silencing” was dependent on the parS sequence and its orientation. The overproduction of P. aeruginosa ParB or its N-terminal part also causes inhibition of the growth of P. aeruginosa and P. putida but not Escherichia coli cells. Since this inhibitory determinant is located well away from ParB segments required for dimerization or interaction with the ParA counterpart, this result may suggest a role for the N terminus of P. aeruginosa ParB in interactions with host cell components.

eps15, a novel tyrosine kinase substrate, exhibits transforming activity

1993 ◽  
Vol 13 (9) ◽  
pp. 5814-5828
Author(s):  
F Fazioli ◽  
L Minichiello ◽  
B Matoskova ◽  
W T Wong ◽  
P P Di Fiore
Keyword(s):  
Gene Product ◽  
Calcium Binding ◽  
Egf Receptor ◽  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Minor Component ◽  
Expression Cloning ◽  
Egfr Signaling Pathway

An expression cloning method which allows direct isolation of cDNAs encoding substrates for tyrosine kinases was applied to the study of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway. A previously undescribed cDNA was isolated and designated eps15. The structural features of the predicted eps15 gene product allow its subdivision into three domains. Domain I contains signatures of a regulatory domain, including a candidate tyrosine phosphorylation site and EF-hand-type calcium-binding domains. Domain II presents the characteristic heptad repeats of coiled-coil rod-like proteins, and domain III displays a repeated aspartic acid-proline-phenylalanine motif similar to a consensus sequence of several methylases. Antibodies specific for the eps15 gene product recognize two proteins: a major species of 142 kDa and a minor component of 155 kDa, both of which are phosphorylated on tyrosine following EGFR activation by EGF in vivo. EGFR is also able to directly phosphorylate the eps15 product in vitro. In addition, phosphorylation of the eps15 gene product in vivo is relatively receptor specific, since the erbB-2 kinase phosphorylates it very inefficiently. Finally, overexpression of eps15 is sufficient to transform NIH 3T3 cells, thus suggesting that the eps15 gene product is involved in the regulation of mitogenic signals.

scholarly journals Retinoblastoma Protein Contains a C-terminal Motif That Targets It for Phosphorylation by Cyclin-cdk Complexes

1999 ◽  
Vol 19 (2) ◽  
pp. 1068-1080 ◽  
Author(s):  
Peter D. Adams ◽  
Xiaotong Li ◽  
William R. Sellers ◽  
Kayla B. Baker ◽  
Xiaohong Leng ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Retinoblastoma Tumor Suppressor Protein ◽  
Retinoblastoma Tumor Suppressor ◽  
C Terminus ◽  
Sequence Elements ◽  
Stable Association ◽  
Retinoblastoma Tumor

ABSTRACT Stable association of certain proteins, such as E2F1 and p21, with cyclin-cdk2 complexes is dependent upon a conserved cyclin-cdk2 binding motif that contains the core sequence ZRXL, where Z and X are usually basic. In vitro phosphorylation of the retinoblastoma tumor suppressor protein, pRB, by cyclin A-cdk2 and cyclin E-cdk2 was inhibited by a short peptide spanning the cyclin-cdk2 binding motif present in E2F1. Examination of the pRB C terminus revealed that it contained sequence elements related to ZRXL. Site-directed mutagenesis of one of these sequences, beginning at residue 870, impaired the phosphorylation of pRB in vitro. A synthetic peptide spanning this sequence also inhibited the phosphorylation of pRB in vitro. pRB C-terminal truncation mutants lacking this sequence were hypophosphorylated in vitro and in vivo despite the presence of intact cyclin-cdk phosphoacceptor sites. Phosphorylation of such mutants was restored by fusion to the ZRXL-like motif derived from pRB or to the ZRXL motifs from E2F1 or p21. Phospho-site-specific antibodies revealed that certain phosphoacceptor sites strictly required a C-terminal ZRXL motif whereas at least one site did not. Furthermore, this residual phosphorylation was sufficient to inactivate pRB in vivo, implying that there are additional mechanisms for directing cyclin-cdk complexes to pRB. Thus, the C terminus of pRB contains a cyclin-cdk interaction motif of the type found in E2F1 and p21 that enables it to be recognized and phosphorylated by cyclin-cdk complexes.

scholarly journals eps15, a novel tyrosine kinase substrate, exhibits transforming activity.

1993 ◽  
Vol 13 (9) ◽  
pp. 5814-5828 ◽  
Author(s):  
F Fazioli ◽  
L Minichiello ◽  
B Matoskova ◽  
W T Wong ◽  
P P Di Fiore
Keyword(s):  
Gene Product ◽  
Calcium Binding ◽  
Egf Receptor ◽  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Minor Component ◽  
Expression Cloning ◽  
Egfr Signaling Pathway

An expression cloning method which allows direct isolation of cDNAs encoding substrates for tyrosine kinases was applied to the study of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway. A previously undescribed cDNA was isolated and designated eps15. The structural features of the predicted eps15 gene product allow its subdivision into three domains. Domain I contains signatures of a regulatory domain, including a candidate tyrosine phosphorylation site and EF-hand-type calcium-binding domains. Domain II presents the characteristic heptad repeats of coiled-coil rod-like proteins, and domain III displays a repeated aspartic acid-proline-phenylalanine motif similar to a consensus sequence of several methylases. Antibodies specific for the eps15 gene product recognize two proteins: a major species of 142 kDa and a minor component of 155 kDa, both of which are phosphorylated on tyrosine following EGFR activation by EGF in vivo. EGFR is also able to directly phosphorylate the eps15 product in vitro. In addition, phosphorylation of the eps15 gene product in vivo is relatively receptor specific, since the erbB-2 kinase phosphorylates it very inefficiently. Finally, overexpression of eps15 is sufficient to transform NIH 3T3 cells, thus suggesting that the eps15 gene product is involved in the regulation of mitogenic signals.

scholarly journals Intestinal Cell Kinase (ICK) Promotes Activation of mTOR Complex 1 (mTORC1) through Phosphorylation of Raptor Thr-908

2012 ◽  
Vol 287 (15) ◽  
pp. 12510-12519 ◽  
Author(s):  
Di Wu ◽  
Jessica R. Chapman ◽  
Lifu Wang ◽  
Thurl E. Harris ◽  
Jeffrey Shabanowitz ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Intestinal Cell ◽  
Putative Phosphorylation Site ◽  
Mtorc1 Signaling ◽  
Mtor Complex 1 ◽  
Intestinal Cell Kinase

Intestinal cell kinase (ICK), named after its cloning origin, the intestine, is actually a ubiquitously expressed and highly conserved serine/threonine protein kinase. Recently we reported that ICK supports cell proliferation and G1 cell cycle progression. ICK deficiency significantly disrupted the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling events. However, the biological substrates that mediate the downstream signaling effects of ICK in proliferation and the molecular mechanisms by which ICK interacts with mTORC1 are not well defined. Our prior studies also provided biochemical evidence that ICK interacts with the mTOR/Raptor complex in cells and phosphorylates Raptor in vitro. In this report, we investigated whether and how ICK targets Raptor to regulate the activity of mTORC1. Using the ICK substrate consensus sequence [R-P-X-S/T-P/A/T/S], we identified a putative phosphorylation site, RPGT908T, for ICK in human Raptor. By mass spectrometry and a phospho-specific antibody, we showed that Raptor Thr-908 is a novel in vivo phosphorylation site. ICK is able to phosphorylate Raptor Thr-908 both in vitro and in vivo and when Raptor exists in protein complexes with or without mTOR. Although expression of the Raptor T908A mutant did not affect the mTORC1 integrity, it markedly impaired the mTORC1 activation by insulin or by overexpression of the small GTP-binding protein RheB under nutrient starvation. Our findings demonstrate an important role for ICK in modulating the activity of mTORC1 through phosphorylation of Raptor Thr-908 and thus implicate a potential signaling mechanism by which ICK regulates cell proliferation and division.

scholarly journals Identification of PNG kinase substrates uncovers interactions with the translational repressor TRAL in the oocyte-to-embryo transition

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Masatoshi Hara ◽  
Sebastian Lourido ◽  
Boryana Petrova ◽  
Hua Jane Lou ◽  
Jessica R Von Stetina ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Phosphorylation Site ◽  
Cyclin B ◽  
Translational Repressor ◽  
Protein Levels ◽  
Site Consensus ◽  
Maternal Mrnas ◽  
Kinase Substrates

The Drosophila Pan Gu (PNG) kinase complex regulates hundreds of maternal mRNAs that become translationally repressed or activated as the oocyte transitions to an embryo. In a previous paper (Hara et al., 2017), we demonstrated PNG activity is under tight developmental control and restricted to this transition. Here, examination of PNG specificity showed it to be a Thr-kinase yet lacking a clear phosphorylation site consensus sequence. An unbiased biochemical screen for PNG substrates identified the conserved translational repressor Trailer Hitch (TRAL). Phosphomimetic mutation of the PNG phospho-sites in TRAL reduced its ability to inhibit translation in vitro. In vivo, mutation of tral dominantly suppressed png mutants and restored Cyclin B protein levels. The repressor Pumilio (PUM) has the same relationship with PNG, and we also show that PUM is a PNG substrate. Furthermore, PNG can phosphorylate BICC and ME31B, repressors that bind TRAL in cytoplasmic RNPs. Therefore, PNG likely promotes translation at the oocyte-to-embryo transition by phosphorylating and inactivating translational repressors.

scholarly journals A Class III PDZ Binding Motif in the Myotilin and FATZ Families Binds Enigma Family Proteins: a Common Link for Z-Disc Myopathies

2008 ◽  
Vol 29 (3) ◽  
pp. 822-834 ◽  
Author(s):  
Pernilla von Nandelstadh ◽  
Mohamed Ismail ◽  
Chiara Gardin ◽  
Heli Suila ◽  
Ivano Zara ◽  
...  
Keyword(s):  
Family Members ◽  
In Vivo Studies ◽  
Binding Motif ◽  
High Homology ◽  
Class Iii ◽  
Pdz Domains ◽  
Muscle Disorders ◽  
C Terminus

ABSTRACT Interactions between Z-disc proteins regulate muscle functions and disruption of these interactions results in muscle disorders. Mutations in Z-disc components myotilin, ZASP/Cypher, and FATZ-2 (calsarcin-1/myozenin-2) are associated with myopathies. We report here that the myotilin and the FATZ (calsarcin/myozenin) families share high homology at their final C-terminal five amino acids. This C-terminal E[ST][DE][DE]L motif is present almost exclusively in these families and is evolutionary conserved. We show by in vitro and in vivo studies that proteins from the myotilin and FATZ (calsarcin/myozenin) families interact via this novel type of class III PDZ binding motif with the PDZ domains of ZASP/Cypher and other Enigma family members: ALP, CLP-36, and RIL. We show that the interactions can be modulated by phosphorylation. Calmodulin-dependent kinase II phosphorylates the C terminus of FATZ-3 (calsarcin-3/myozenin-3) and myotilin, whereas PKA phosphorylates that of FATZ-1 (calsarcin-2/myozenin-1) and FATZ-2 (calsarcin-1/myozenin-1). This is the first report of a binding motif common to both the myotilin and the FATZ (calsarcin/myozenin) families that is specific for interactions with Enigma family members.

scholarly journals The C Terminus of the Histone Chaperone Asf1 Cross-Links to Histone H3 in Yeast and Promotes Interaction with Histones H3 and H4

2012 ◽  
Vol 33 (3) ◽  
pp. 605-621 ◽  
Author(s):  
Briana K. Dennehey ◽  
Seth Noone ◽  
Wallace H. Liu ◽  
Luke Smith ◽  
Mair E. A. Churchill ◽  
...  
Keyword(s):  
Amino Acid ◽  
Phosphorylation Site ◽  
Histone H3 ◽  
Unnatural Amino Acid ◽  
Full Length ◽  
Content Type ◽  
C Terminus ◽  
Cross Links

ABSTRACTThe central histone H3/H4 chaperone Asf1 comprises a highly conserved globular core and a divergent C-terminal tail. While the function and structure of the Asf1 core are well known, the function of the tail is less well understood. Here, we have explored the role of the yeast (yAsf1) and human (hAsf1a and hAsf1b) Asf1 tails inSaccharomyces cerevisiae. We show, using a photoreactive, unnatural amino acid, that Asf1 tail residue 210 cross-links to histone H3in vivoand, further, that loss of C-terminal tail residues 211 to 279 weakens yAsf1-histone binding affinityin vitronearly 200-fold. Via several yAsf1 C-terminal truncations and yeast-human chimeric proteins, we found that truncations at residue 210 increase transcriptional silencing and that the hAsf1a tail partially substitutes for full-length yAsf1 with respect to silencing but that full-length hAsf1b is a better overall substitute for full-length yAsf1. In addition, we show that the C-terminal tail of Asf1 is phosphorylated at T270 in yeast. Loss of this phosphorylation site does not prevent coimmunoprecipitation of yAsf1 and Rad53 from yeast extracts, whereas amino acid residue substitutions at the Asf1-histone H3/H4 interface do. Finally, we show that residue substitutions in yAsf1 near the CAF-1/HIRA interface also influence yAsf1's function in silencing.

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