scholarly journals Selective loss of substrate recognition induced by the tumour-associated D294G point mutation in protein kinase Cα

1998 ◽  
Vol 334 (2) ◽  
pp. 393-397 ◽  
Author(s):  
Corinne PRÉVOSTEL ◽  
Véronique ALVARO ◽  
Alice VALLENTIN ◽  
Annick MARTIN ◽  
Susan JAKEN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Protein Interactions ◽  
Kinase Activity ◽  
Substrate Recognition ◽  
Mutant Enzyme ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Selective Loss ◽  
Protein Kinase Cα

The tumour-associated D294G mutant of protein kinase Cα (PKCα) was recently shown not to be translocated to the plasma membrane on stimulation with PMA, in contrast with the wild-type enzyme. Using recombinant wild-type and mutant PKCα, we establish here that, although the PKCα intrinsic lipid-dependent catalytic activity remains unaltered by the D294G mutation, the mutant enzyme exhibits a selective loss of substrate recognition. Indeed, whereas the mutant enzyme is still able to phosphorylate histone IIIS with comparable efficiency to that of the wild-type enzyme, it exhibits a lack of kinase activity towards the previously cloned 35F and 35H substrates for PKC. Overlay experiments demonstrate that this selective loss of kinase activity is correlated with a decrease in binding of D294G PKCα to the 35F and 35H proteins compared with that of the wild-type enzyme. Because the 35H and 35F proteins are predicted to be PKCα-anchoring proteins, these findings suggest a selective loss of PKCα–protein interactions that might fail to stabilize the location of the PKCα mutant at the plasma membrane.

scholarly journals A Single Point Mutation in the V3 Region Affects Protein Kinase Cα Targeting and Accumulation at Cell-Cell Contacts

2001 ◽  
Vol 21 (10) ◽  
pp. 3351-3363 ◽  
Author(s):  
Alice Vallentin ◽  
Thi-Chang Lo ◽  
Dominique Joubert
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Single Point ◽  
Single Point Mutation ◽  
Actin Network ◽  
Wild Type ◽  
Cell Contacts ◽  
Regulatory Processes ◽  
Protein Kinase Cα ◽  
Cell Cell

ABSTRACT Given the importance of intercellular adhesion for many regulatory processes, we have investigated the control of protein kinase Cα (PKCα) targeting to the cell-cell contacts. We have previously shown that, upon treatment of the pituitary cell line GH3B6 with thyrotropin-releasing hormone (TRH) or phorbol 12-myristate 13-acetate (PMA), human PKCα (hPKCα) is selectively targeted to the cell-cell contacts (42). Here we show that the D294G mutation of hPKCα, previously identified in a subpopulation of human tumors, induces the loss of this selective targeting. The D294G mutant is instead targeted to the entire plasma membrane, including the cell-cell contacts, and the duration of the first rapid and transient translocation induced by TRH (42) is longer than that of the wild-type enzyme (93.3 versus 22.5 s), coinciding with the duration of the [Ca2+]i increase. We found that in the presence or absence of PMA, RACK1 is never localized at the cell-cell contacts nor was it coimmunoprecipitated with hPKCα wild type or the D294G mutant. In contrast, PMA treatment or long-term TRH stimulation resulted in the presence of F-actin and β-catenin at the cell-cell contacts and their exclusion from the rest of the plasma membrane. Upon disruption of the F-actin network with phalloidin or cytochalasin D, wild-type hPKCα translocates but did not accumulate at the plasma membrane and β-catenin did not accumulate at the cell-cell contacts. In contrast, the disruption of the F-actin network affected neither translocation nor accumulation of the D294G mutant. These results show that the presence of PKCα at the cell-cell contacts is a regulated process which depends upon the integrity of both PKCα and the actin microfilament network.

PROTEIN KINASE ACTIVITY ASSOCIATED WITH THE FAT CELL PLASMA MEMBRANE

1981 ◽  
Vol 9 (2) ◽  
pp. 232P-232P
Author(s):  
G. J. Belsham ◽  
R. W. Brownsey ◽  
R. M. Denton
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Fat Cell ◽  
Cell Plasma Membrane ◽  
Cell Plasma

scholarly journals A Regulator of G Protein Signaling-containing Kinase Is Important for Chemotaxis and Multicellular Development inDictyostelium

2003 ◽  
Vol 14 (4) ◽  
pp. 1727-1743 ◽  
Author(s):  
Binggang Sun ◽  
Richard A. Firtel
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Kinase Activity ◽  
Site Directed Mutagenesis ◽  
Membrane Localization ◽  
Pleckstrin Homology Domain ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Wild Type ◽  
Gene Encoding

We have identified a gene encoding RGS domain-containing protein kinase (RCK1), a novel regulator of G protein signaling domain-containing protein kinase. RCK1 mutant strains exhibit strong aggregation and chemotaxis defects. rck1 null cells chemotax ∼50% faster than wild-type cells, suggesting RCK1 plays a negative regulatory role in chemotaxis. Consistent with this finding, overexpression of wild-type RCK1 reduces chemotaxis speed by ∼40%. On cAMP stimulation, RCK1 transiently translocates to the membrane/cortex region with membrane localization peaking at ∼10 s, similar to the kinetics of membrane localization of the pleckstrin homology domain-containing proteins CRAC, Akt/PKB, and PhdA. RCK1 kinase activity also increases dramatically. The RCK1 kinase activity does not rapidly adapt, but decreases after the cAMP stimulus is removed. This is particularly novel considering that most other chemoattractant-activated kinases (e.g., Akt/PKB, ERK1, ERK2, and PAKa) rapidly adapt after activation. Using site-directed mutagenesis, we further show that both the RGS and kinase domains are required for RCK1 function and that RCK1 kinase activity is required for the delocalization of RCK1 from the plasma membrane. Genetic evidence suggests RCK1 function lies downstream from Gα2, the heterotrimeric G protein that couples to the cAMP chemoattractant receptors. We suggest that RCK1 might be part of an adaptation pathway that regulates aspects of chemotaxis in Dictyostelium.

Regulation of rat Na+-K+-ATPase activity by PKC is modulated by state of phosphorylation of Ser-943 by PKA

1997 ◽  
Vol 273 (6) ◽  
pp. C1981-C1986 ◽  
Author(s):  
Xian-Jun Cheng ◽  
Jan-Olov Höög ◽  
Angus C. Nairn ◽  
Paul Greengard ◽  
Anita Aperia
Keyword(s):  
Enzyme Activity ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Response To Treatment ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Present Evidence ◽  
Cos Cells ◽  
Kinase C ◽  
Kinase A

We have previously shown that the rat Na+-K+-ATPase α1-isoform is phosphorylated at Ser-943 by protein kinase A (PKA) and at Ser-23 by protein kinase C (PKC), which in both cases results in inhibition of enzyme activity. We now present evidence that suggests that the phosphorylation of Ser-943 by PKA modulates the response of Na+-K+-ATPase to PKC. Rat Na+-K+-ATPase α1 or a mutant in which Ser-943 was changed to Ala-943 was stably expressed in COS cells. The inhibition of enzyme activity measured in response to treatment with the phorbol ester, phorbol 12,13-dibutyrate (PDBu; 10−6 M), was significantly reduced in the cells expressing the Ala-943 mutant compared with that observed in cells expressing wild-type enzyme. In contrast, for cells expressing Na+-K+-ATPase α1 in which Ser-943 was mutated to Asp-943, the effect of PDBu was slightly enhanced. The PDBu-induced inhibition was not mediated by activation of the adenosine 3′,5′-cyclic monophosphate/PKA system and was not achieved via direct phosphorylation of Ser-943. Sp-5,6-DCl-cBIMPS, a specific PKA activator, increased the phosphorylation of Ser-943, and this was associated with an enhanced response to PDBu. Thus the effect of PKC on rat Na+-K+-ATPase α1 is determined not only by the activity of PKC but also by the state of phosphorylation of Ser-943.

Suppression of Synaptotagmin II restrains phorbolester-induced downregulation of protein kinase Cα by diverting the kinase from a degradative pathway to the recycling endocytic compartment

2002 ◽  
Vol 115 (15) ◽  
pp. 3083-3092 ◽  
Author(s):  
Ze Peng ◽  
Elena Grimberg ◽  
Ronit Sagi-Eisenberg
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Secretory Granules ◽  
Critical Factor ◽  
Cellular Level ◽  
Endocytic Compartment ◽  
Early Endosomes ◽  
Protein Kinase Cα ◽  
Rbl Cells

Downregulation of protein kinase Cα (PKCα) following long-term exposure to phorbol esters such as TPA is traffic dependent and involves delivery of the active, membrane-associated PKCα to endosomes. In this study, we show that synaptotagmin II (Syt II), a member of the Syt family of proteins, is required for TPA-induced degradation of PKCα. Thus, whereas the kinase half-life in TPA-treated cultured mast cells (the mast cell line rat basophilic leukemia RBL-2H3) is 2 hours, it is doubled in RBL-Syt II- cells, in which the cellular level of Syt II is reduced by>95% by transfection with Syt II antisense cDNA. We demonstrate that in TPA-treated RBL cells, PKCα travels from the cytosol to the plasma membrane, where it is delivered to early endosomes on its route to degradation. By contrast, in TPA-treated RBL-Syt II- cells,PKCα is diverted to recycling endosomes and remains distributed between the plasma membrane and the perinuclear recycling endocytic compartment. Notably, in both RBL and RBL-Syt II- cells, a fraction of PKCα is delivered and maintained in the secretory granules (SG). These results implicate Syt II as a critical factor for the delivery of internalized cargo for degradation. As shown here, one consequence of Syt II suppression is a delay in PKCα downregulation, resulting in its prolonged signaling.

scholarly journals Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein.

1993 ◽  
Vol 13 (5) ◽  
pp. 2899-2908 ◽  
Author(s):  
A L Jackson ◽  
P M Pahl ◽  
K Harrison ◽  
J Rosamond ◽  
R A Sclafani
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Protein Interactions ◽  
Kinase Activity ◽  
Mitotic Cell ◽  
Common Point ◽  
Temperature Sensitive ◽  
Cdc7 Kinase

Yeast Cdc7 protein kinase and Dbf4 protein are both required for the initiation of DNA replication at the G1/S phase boundary of the mitotic cell cycle. Cdc7 kinase function is stage-specific in the cell cycle, but total Cdc7 protein levels remained unchanged. Therefore, regulation of Cdc7 function appears to be the result of posttranslational modification. In this study, we have attempted to elucidate the mechanism responsible for achieving this specific execution point of Cdc7. Cdc7 kinase activity was shown to be maximal at the G1/S boundary by using either cultures synchronized with alpha factor or Cdc- mutants or with inhibitors of DNA synthesis or mitosis. Therefore, Cdc7 kinase is regulated by a posttranslational mechanism that ensures maximal Cdc7 activity at the G1/S boundary, which is consistent with Cdc7 function in the cell cycle. This cell cycle-dependent regulation could be the result of association with the Dbf4 protein. In this study, the Dbf4 protein was shown to be required for Cdc7 kinase activity in that Cdc7 kinase activity is thermolabile in vitro when extracts prepared from a temperature-sensitive dbf4 mutant grown under permissive conditions are used. In vitro reconstitution assays, in addition to employment of the two-hybrid system for protein-protein interactions, have demonstrated that the Cdc7 and Dbf4 proteins interact both in vitro and in vivo. A suppressor mutation, bob1-1, which can bypass deletion mutations in both cdc7 and dbf4 was isolated. However, the bob1-1 mutation cannot bypass all events in G1 phase because it fails to suppress temperature-sensitive cdc4 or cdc28 mutations. This indicates that the Cdc7 and Dbf4 proteins act at a common point in the cell cycle. Therefore, because of the common point of function for the two proteins and the fact that the Dbf4 protein is essential for Cdc7 function, we propose that Dbf4 may represent a cyclin-like molecule specific for the activation of Cdc7 kinase.

scholarly journals Structural identification of the myo-inositol 1,4,5-trisphosphate-binding domain in rat brain inositol 1,4,5-trisphosphate 3-kinase

1997 ◽  
Vol 326 (1) ◽  
pp. 221-225 ◽  
Author(s):  
Shinji TOGASHI ◽  
Kazunaga TAKAZAWA ◽  
Toyoshi ENDO ◽  
Christophe ERNEUX ◽  
Toshimasa ONAYA
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Apparent Homogeneity ◽  
Inositol 1,4,5 Trisphosphate ◽  
Charged Amino Acid Residue

A series of key amino acids involved in Ins(1,4,5)P3 (InsP3) binding and catalytic activity of rat brain InsP3 3-kinase has been identified. The catalytic domain is at the C-terminal end and restricted to a maximum of 275 amino acids [Takazawa and Erneux (1991) Biochem. J. 280, 125–129]. In this study, newly prepared 5′-deletion and site-directed mutants have been compared both for InsP3 binding and InsP3 3-kinase activity. When the protein was expressed from L259 to R459, the activity was lost but InsP3 binding was conserved. Another deletion mutant that had lost only four amino acids after L259 had lost InsP3 binding, and this finding suggests that these residues (i.e. L259DCK262) are involved in InsP3 binding. To further support the data, we have produced two mutants by site-directed mutagenesis on residues C261 and K262. The two new enzymes were designated M4 (C261S) and M5 (K262A). M4 showed similar Vmax and Km values for InsP3 and ATP to wild-type enzyme. In contrast, M5 was totally inactive but had kept the ability to bind to calmodulin–Sepharose. C-terminal deletion mutants that had lost five, seven or nine amino acids showed a large decrease in InsP3 binding and InsP3 3-kinase activity. One mutant that had lost five amino acids (M2) was purified to apparent homogeneity: Km values for both substrates appeared unchanged but Vmax was decreased approx. 40-fold compared with the wild-type enzyme. The results indicate that (1) a positively charged amino acid residue K262 is essential for InsP3 binding and (2) amino acids at the C-terminal end of the protein are necessary to act as a catalyst in the InsP3 3-kinase reaction.

A Wild-Type Nanopore Sensor for Protein Kinase Activity

2019 ◽  
Vol 91 (15) ◽  
pp. 9910-9915 ◽  
Author(s):  
Fu-Na Meng ◽  
Yi-Lun Ying ◽  
Jie Yang ◽  
Yi-Tao Long
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Wild Type
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