scholarly journals Retargeting of cytosolic proteins to the plasma membrane by the Lck protein tyrosine kinase dual acylation motif

1997 ◽  
Vol 110 (5) ◽  
pp. 673-679 ◽  
Author(s):  
P. Zlatkine ◽  
B. Mehul ◽  
A.I. Magee
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Subcellular Fractionation ◽  
Cytosolic Proteins ◽  
Protein Tyrosine ◽  
Confocal Immunofluorescence Microscopy ◽  
Ionic Detergent ◽  
Galectin 3 ◽  
Lck Protein

Several members of the Src family of protein tyrosine kinases have a N-terminal dual acylation motif which specifies their myristoylation and S-acylation. These lipid modifications are necessary for correct intracellular localisation to the plasma membrane and to detergent-resistant glycolipid-enriched membrane domains (GEMs). Using chimaeras of the Lck dual acylation motif with two normally cytosolic proteins (chloramphenicol acetyl transferase and galectin-3), we show here that this motif is sufficient to encode correct lipid modification and to target these chimaeras to the plasma membrane, as demonstrated by subcellular fractionation and confocal immunofluorescence microscopy of transiently transfected COS cells. In addition, the chimaeras are resistant to extraction with cold non-ionic detergent, indicating targeting to GEM subdomains in the plasma membrane. The dual acylation motif has potential for targeting proteins to specific plasma membrane subdomains involved in signalling.

scholarly journals Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes

1999 ◽  
Vol 10 (4) ◽  
pp. 891-905 ◽  
Author(s):  
Subburaj Ilangumaran ◽  
Stephan Arni ◽  
Gerhild van Echten-Deckert ◽  
Bettina Borisch ◽  
Daniel C. Hoessli
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Plasma Membranes ◽  
Tyrosine Phosphatase ◽  
Transmembrane Proteins ◽  
Subcellular Fractionation ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
Nonionic Detergents

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.

scholarly journals Myristoylation and differential palmitoylation of the HCK protein-tyrosine kinases govern their attachment to membranes and association with caveolae.

1995 ◽  
Vol 15 (7) ◽  
pp. 3507-3515 ◽  
Author(s):  
S M Robbins ◽  
N A Quintrell ◽  
J M Bishop
Keyword(s):  
Fatty Acids ◽  
Plasma Membrane ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
Lipid Modifications ◽  
Molecular Weights ◽  
Protein Palmitoylation

The human proto-oncogene HCK encodes two versions of a protein-tyrosine kinase, with molecular weights of 59,000 (p59hck) and 61,000 (p61hck). The two proteins arise from a single mRNA by alternative initiations of translation. In this study, we explored the functions of these proteins by determining their locations within cells and by characterizing lipid modifications required for the proteins to reach those locations. We found that p59hck is entirely associated with cellular membranes, including the organelles known as caveolae; in contrast, only a portion of p61hck is situated on membranes, and none is detectable in preparations of caveolae. These distinctions can be attributed to differential modification of the two HCK proteins with fatty acids. Both proteins are at least in part myristoylated, p59hck more so than p61hck. In addition, however, p59hck is palmitoylated on cysteine 3 in the protein. Palmitoylation of the protein requires prior myristoylation and, in turn, is required for targeting to caveolae. These findings are in accord with recent reports for other members of the SRC family of protein-tyrosine kinases. Taken together, the results suggest that HCK and several of its relatives may participate in the functions of caveolae, which apparently include the transduction of signals across the plasma membrane to the interior of the cell.

scholarly journals Development of the Sensing Platform for Protein Tyrosine Kinase Activity

Biosensors ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 240
Author(s):  
Lan-Yi Wei ◽  
Wei Lin ◽  
Bey-Fen Leo ◽  
Lik-Voon Kiew ◽  
Chia-Ching Chang ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Significant Inhibition ◽  
Tyrosine Kinase Activity ◽  
Protein Tyrosine ◽  
Sensing Platform ◽  
Relative Standard ◽  
Protein Tyrosine Kinase Activity

A miniature tyrosinase-based electrochemical sensing platform for label-free detection of protein tyrosine kinase activity was developed in this study. The developed miniature sensing platform can detect the substrate peptides for tyrosine kinases, such as c-Src, Hck and Her2, in a low sample volume (1–2 μL). The developed sensing platform exhibited a high reproducibility for repetitive measurement with an RSD (relative standard deviation) of 6.6%. The developed sensing platform can detect the Hck and Her2 in a linear range of 1–200 U/mL with the detection limit of 1 U/mL. The sensing platform was also effective in assessing the specificity and efficacies of the inhibitors for protein tyrosine kinases. This is demonstrated by the detection of significant inhibition of Hck (~88.1%, but not Her2) by the Src inhibitor 1, an inhibitor for Src family kinases, as well as the significant inhibition of Her2 (~91%, but not Hck) by CP-724714 through the platform. These results suggest the potential of the developed miniature sensing platform as an effective tool for detecting different protein tyrosine kinase activity and for accessing the inhibitory effect of various inhibitors to these kinases.

Protein tyrosine kinase signaling is necessary for NO donor-induced late preconditioning against myocardial stunning

2003 ◽  
Vol 284 (4) ◽  
pp. H1441-H1448 ◽  
Author(s):  
Xian-Liang Tang ◽  
Eitaro Kodani ◽  
Hitoshi Takano ◽  
Michael Hill ◽  
Ken Shinmura ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Myocardial Stunning ◽  
Protein Tyrosine Kinase ◽  
Late Phase ◽  
Recovery Period ◽  
Wall Thickening ◽  
No Donor ◽  
Signaling Mechanism ◽  
Protein Tyrosine ◽  
Conscious Rabbits

Although protein tyrosine kinases (PTKs) signaling has been implicated in the late phase of ischemic preconditioning (PC), it is unknown whether PTK signaling is necessary for the development of nitric oxide (NO) donor-induced late PC. Thus conscious rabbits underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles followed by a 5-h recovery period of reperfusion for 3 consecutive days ( days 1, 2, and 3). On day 0 (24 h before the 6 O/R cycles on day 1), rabbits received no treatment (control), the NO donor diethylenetriamine (DETA)/NO (DETA/NO), the PTK inhibitor 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4- d]pyrimidine (PP2), or DETA/NO plus PP2 (DETA/NO + PP2). In control rabbits ( n = 6), the six O/R cycles on day 1resulted in delayed functional recovery, indicating severe myocardial stunning. In rabbits pretreated with DETA/NO ( n = 5) on day 1, myocardial stunning caused by the six O/R cycles on day 1 was markedly attenuated, with a significant reduction (∼60%) in the total deficit of wall thickening (WTh) compared with controls, indicating that DETA/NO induced a late PC effect against stunning. However, in rabbits pretreated with DETA/NO + PP2 ( n = 5), the total deficit of WTh was significantly greater than that in rabbits treated with DETA/NO alone and was similar to that in controls, indicating that PP2 prevented the development of DETA/NO-induced late PC. In rabbits pretreated with PP2 on day 0 ( n = 4), the total deficit of WTh was similar to that in controls, indicating that PP2 does not affect myocardial stunning in itself. We conclude that a PTK-dependent signaling mechanism is necessary for the development of NO donor-induced late PC against myocardial stunning in conscious rabbits.

scholarly journals Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains.

1995 ◽  
Vol 130 (5) ◽  
pp. 1181-1187 ◽  
Author(s):  
M D Schaller ◽  
C A Otey ◽  
J D Hildebrand ◽  
J T Parsons
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Cytoplasmic Domain ◽  
Embryo Cell ◽  
Vitro Assay ◽  
Protein Tyrosine ◽  
Signaling Complex

The integrins have recently been implicated in signal transduction. A likely mediator of integrin signaling is focal adhesion kinase (pp125FAK or FAK), a structurally distinct protein tyrosine kinase that becomes enzymatically activated upon engagement of integrins with their ligands. A second candidate signaling molecule is paxillin, a focal adhesion associated, cytoskeletal protein that coordinately becomes phosphorylated on tyrosine upon activation of pp125FAK. Paxillin physically complexes with two protein tyrosine kinases, pp60src and Csk (COOH-terminal src kinase), and the oncoprotein p47gag-crk, each of which could function as part of a paxillin signaling complex. Using an in vitro assay we have established that the cytoplasmic domain of the beta 1 integrin can bind to paxillin and pp125FAK from chicken embryo cell lysates. The NH2-terminal, noncatalytic domain of pp125FAK can bind directly to the cytoplasmic tail of beta 1 and recognizes integrin sequences distinct from those involved in binding to alpha-actinin. Paxillin binding is independent of pp125FAK binding despite the fact that both bind to the same region of beta 1. These results demonstrate that the cytoplasmic domain of the beta subunits of integrins contain binding sites for both signaling molecules and structural proteins suggesting that integrins can coordinate the generation of cytoplasmic signals in addition to their role in anchoring components of the cytoskeleton.

The myristylation signal of p60v-src functionally complements the N-terminal fps-specific region of P130gag-fps

1989 ◽  
Vol 9 (5) ◽  
pp. 2214-2219
Author(s):  
A R Brooks-Wilson ◽  
E Ball ◽  
T Pawson
Keyword(s):  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Specific Region ◽  
Protein Tyrosine Kinases ◽  
Membrane Association ◽  
Modular Construction ◽  
Specific Domain ◽  
Protein Tyrosine ◽  
Sarcoma Virus ◽  
Transforming Activity

The P130gag-fps protein-tyrosine kinase of Fujinami sarcoma virus contains an N-terminal fps-specific domain (Nfps) that is important for oncogenicity. The N-terminal 14 amino acids of p60v-src, which direct myristylation and membrane association, can replace the gag-Nfps sequences of P130gag-fps (residues 1 to 635), producing a highly transforming src-fps polypeptide. Conversely, gag-Nfps can restore modest transforming activity to a nonmyristylated v-src polypeptide. These results emphasize the modular construction of protein-tyrosine kinases and indicate that Nfps, possibly in conjunction with gag, functions in the subcellular localization of P130gag-fps.

Functional dissection of p56lck, a protein tyrosine kinase which mediates interleukin-2-induced activation of the c-fos gene

1994 ◽  
Vol 14 (9) ◽  
pp. 5812-5819
Author(s):  
H Shibuya ◽  
K Kohu ◽  
K Yamada ◽  
E L Barsoumian ◽  
R M Perlmutter ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Gene Activation ◽  
Interleukin 2 ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Amino Acid Residues ◽  
Protein Tyrosine ◽  
Responsive Element

Members of the newly identified receptor family for cytokines characteristically lack the intrinsic protein tyrosine kinase domain that is a hallmark of other growth factor receptors. Instead, accumulating evidence suggests that these receptors utilize nonreceptor-type protein tyrosine kinases for downstream signal transduction by cytokines. We have shown previously that the interleukin-2 receptor beta-chain interacts both physically and functionally with a Src family member, p56lck, and that p56lck activation leads to induction of the c-fos gene. However, the mechanism linking p56lck activation with c-fos induction remains unelucidated. In the present study, we systematically examined the extent of c-fos promoter activation by expression of a series of p56lck mutants, using a transient cotransfection assay. The results define a set of the essential amino acid residues that regulate p56lck induction of the c-fos promoter. We also provide evidence that the serum-responsive element and sis-inducible element are both targets through which p56lck controls c-fos gene activation.

Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK

1994 ◽  
Vol 14 (1) ◽  
pp. 147-155
Author(s):  
B S Cobb ◽  
M D Schaller ◽  
T H Leu ◽  
J T Parsons
Keyword(s):  
Focal Adhesion ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Src Family Kinases ◽  
Protein Tyrosine Kinases ◽  
Protein Tyrosine ◽  
Sh2 Domains ◽  
Embryo Cells ◽  
Stable Association

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.

Characterization of elk, a brain-specific receptor tyrosine kinase

1991 ◽  
Vol 11 (5) ◽  
pp. 2496-2502
Author(s):  
V Lhoták ◽  
P Greer ◽  
K Letwin ◽  
T Pawson
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Catalytic Domain ◽  
Signal Sequence ◽  
Tyrosine Kinase Domain ◽  
Rat Tissues ◽  
Protein Tyrosine

The elk gene encodes a novel receptorlike protein-tyrosine kinase, which belongs to the eph subfamily. We have previously identified a partial cDNA encompassing the elk catalytic domain (K. Letwin, S.-P. Yee, and T. Pawson, Oncogene 3:621-678, 1988). Using this cDNA as a probe, we have isolated cDNAs spanning the entire rat elk coding sequence. The predicted Elk protein contains all the hallmarks of a receptor tyrosine kinase, including an N-terminal signal sequence, a cysteine-rich extracellular domain, a membrane-spanning segment, a cytoplasmic tyrosine kinase domain, and a C-terminal tail. In both amino acid sequence and overall structure, Elk is most similar to the Eph and Eck protein-tyrosine kinases, suggesting that the eph, elk, and eck genes encode members of a new subfamily of receptorlike tyrosine kinases. Among rat tissues, elk expression appears restricted to brain and testes, with the brain having higher levels of both elk RNA and protein. Elk protein immunoprecipitated from a rat brain lysate becomes phosphorylated on tyrosine in an in vitro kinase reaction, consistent with the prediction that the mammalian elk gene encodes a tyrosine kinase capable of autophosphorylation. The characteristics of the Elk tyrosine kinase suggest that it may be involved in cell-cell interactions in the nervous system.

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