scholarly journals Phosphorylation-dependent Localization of Microtubule-associated Protein MAP2c to the Actin Cytoskeleton

2000 ◽  
Vol 11 (10) ◽  
pp. 3573-3587 ◽  
Author(s):  
Rachel S. Ozer ◽  
Shelley Halpain
Keyword(s):  
Protein Kinase ◽  
Actin Cytoskeleton ◽  
Hela Cells ◽  
Protein Kinase Activity ◽  
Homologous Proteins ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Microtubule-associated protein 2 (MAP2) is a neuronal phosphoprotein that promotes net microtubule growth and actin cross-linking and bundling in vitro. Little is known about MAP2 regulation or its interaction with the cytoskeleton in vivo. Here we investigate the in vivo function of three specific sites of phosphorylation on MAP2. cAMP-dependent protein kinase activity disrupts the MAP2–microtubule interaction in living HeLa cells and promotes MAP2c localization to peripheral membrane ruffles enriched in actin. cAMP-dependent protein kinase phosphorylates serines within three KXGS motifs, one within each tubulin-binding repeat. These highly conserved motifs are also found in homologous proteins tau and MAP4. Phosphorylation at two of these sites was detected in brain tissue. Constitutive phosphorylation at these sites was mimicked by single, double, and triple mutations to glutamic acid. Biochemical and microscopy-based assays indicated that mutation of a single residue was adequate to disrupt the MAP2–microtubule interaction in HeLa cells. Double or triple point mutation promoted MAP2c localization to the actin cytoskeleton. Specific association between MAP2c and the actin cytoskeleton was demonstrated by retention of MAP2c–actin colocalization after detergent extraction. Specific phosphorylation states may enhance the interaction of MAP2 with the actin cytoskeleton, thereby providing a regulated mechanism for MAP2 function within distinct cytoskeletal domains.

scholarly journals The DNA-Dependent Protein Kinase Catalytic Subunit Is Phosphorylated In Vivo on Threonine 3950, a Highly Conserved Amino Acid in the Protein Kinase Domain

2006 ◽  
Vol 27 (5) ◽  
pp. 1581-1591 ◽  
Author(s):  
Pauline Douglas ◽  
Xiaoping Cui ◽  
Wesley D. Block ◽  
Yaping Yu ◽  
Shikha Gupta ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Kinase Domain ◽  
Catalytic Subunit ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Protein Kinase Domain ◽  
Dependent Protein

ABSTRACT The protein kinase activity of the DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs) via the process of nonhomologous end joining (NHEJ). However, to date, the only target shown to be functionally relevant for the enzymatic role of DNA-PK in NHEJ is the large catalytic subunit DNA-PKcs itself. In vitro, autophosphorylation of DNA-PKcs induces kinase inactivation and dissociation of DNA-PKcs from the DNA end-binding component Ku70/Ku80. Phosphorylation within the two previously identified clusters of phosphorylation sites does not mediate inactivation of the assembled complex and only partially regulates kinase disassembly, suggesting that additional autophosphorylation sites may be important for DNA-PK function. Here, we show that DNA-PKcs contains a highly conserved amino acid (threonine 3950) in a region similar to the activation loop or t-loop found in the protein kinase domain of members of the typical eukaryotic protein kinase family. We demonstrate that threonine 3950 is an in vitro autophosphorylation site and that this residue, as well as other previously identified sites in the ABCDE cluster, is phosphorylated in vivo in irradiated cells. Moreover, we show that mutation of threonine 3950 to the phosphomimic aspartic acid abrogates V(D)J recombination and leads to radiation sensitivity. Together, these data suggest that threonine 3950 is a functionally important, DNA damage-inducible phosphorylation site and that phosphorylation of this site regulates the activity of DNA-PKcs.

scholarly journals Identification of in vitro and in vivo phosphorylation sites in the catalytic subunit of the DNA-dependent protein kinase

2002 ◽  
Vol 368 (1) ◽  
pp. 243-251 ◽  
Author(s):  
Pauline DOUGLAS ◽  
Gopal P. SAPKOTA ◽  
Nick MORRICE ◽  
Yaping YU ◽  
Aaron A. GOODARZI ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Okadaic Acid ◽  
Kinase Activity ◽  
Catalytic Subunit ◽  
Protein Sequencing ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs), such as those caused by ionizing radiation and other DNA-damaging agents. DNA-PK is composed of a large catalytic subunit (DNA-PKcs) and a heterodimer of Ku70 and Ku80 that assemble on the ends of double-stranded DNA to form an active serine/threonine protein kinase complex. Despite in vitro and in vivo evidence to support an essential role for the protein kinase activity of DNA-PK in the repair of DNA DSBs, the physiological targets of DNA-PK have remained elusive. We have previously shown that DNA-PK undergoes autophosphorylation in vitro, and that autophosphorylation correlates with loss of protein kinase activity and dissociation of the DNA-PK complex. Also, treatment of cells with the protein phosphatase inhibitor, okadaic acid, enhances DNA-PKcs phosphorylation and reduces DNA-PK activity in vivo. Here, using solid-phase protein sequencing, MS and phosphospecific antibodies, we have identified seven in vitro autophosphorylation sites in DNA-PKcs. Six of these sites (Thr2609, Ser2612, Thr2620, Ser2624, Thr2638 and Thr2647) are clustered in a region of 38 amino acids in the central region of the protein. Five of these sites (Thr2609, Ser2612, Thr2638, Thr2647 and Ser3205) are conserved between six vertebrate species. Moreover, we show that DNA-PKcs is phosphorylated in vivo at Thr2609, Ser2612, Thr2638 and Thr2647 in okadaic acid-treated human cells. We propose that phosphorylation of these sites may play an important role in DNA-PK function.

scholarly journals Hydralazine targets cAMP-dependent protein kinase leading to sirtuin1/5 activation and lifespan extension in C. elegans

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Esmaeil Dehghan ◽  
Mohammad Goodarzi ◽  
Bahar Saremi ◽  
Rueyling Lin ◽  
Hamid Mirzaei
Keyword(s):  
Protein Kinase ◽  
Mitochondrial Function ◽  
Dependent Protein Kinase ◽  
In Vivo Models ◽  
C Elegans ◽  
Beneficial Effects ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Abstract Therapeutic activation of mitochondrial function has been suggested as an effective strategy to combat aging. Hydralazine is an FDA-approved drug used in the treatment of hypertension, heart failure and cancer. Hydralazine has been recently shown to promote lifespan in C. elegans, rotifer and yeast through a mechanism which has remained elusive. Here we report cAMP-dependent protein kinase (PKA) as the direct target of hydralazine. Using in vitro and in vivo models, we demonstrate a mechanism in which binding and stabilization of a catalytic subunit of PKA by hydralazine lead to improved mitochondrial function and metabolic homeostasis via the SIRT1/SIRT5 axis, which underlies hydralazine’s prolongevity and stress resistance benefits. Hydralazine also protects mitochondrial metabolism and function resulting in restoration of health and lifespan in C. elegans under high glucose and other stress conditions. Our data also provide new insights into the mechanism(s) that explain various other known beneficial effects of hydralazine.

PHOSPHORYLATION IN VITRO OF ADRENAL SUBCELLULAR PROTEINS BY ADENOSINE-3′, 5′-MONOPHOSPHATE-DEPENDENT PROTEIN KINASE: EFFECT OF ACTH IN VIVO

1974 ◽  
Vol 75 (2) ◽  
pp. 325-332 ◽  
Author(s):  
Shogo Ichii ◽  
Noriko Murakami ◽  
Akemi Ikeda
Keyword(s):  
Protein Kinase ◽  
Untreated Control ◽  
Adrenal Glands ◽  
Dependent Protein Kinase ◽  
Good Substrate ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Endogenous Substrates

ABSTRACT In vitro phosphorylation of protein from rat adrenal subcellular fractions by partially purified cAMP-dependent protein kinase isolated from rat adrenal and liver was investigated. Only proteins from microsomes were found to be a good substrate for the enzyme. Rates of phosphorylation of adrenal microsomal and cytosol proteins isolated from animals which received ACTH were significantly lower than those from untreated control animals. The results obtained in this study seem to indicate that the microsomal proteins are one of the major endogenous substrates of the cAMP-dependent protein kinase in rat adrenal glands.

scholarly journals Differential Effects of Carcinogens on Hepatic Cytosolic Cyclic AMP-dependent Protein Kinase Activity

1986 ◽  
Vol 5 (4) ◽  
pp. 267-273
Author(s):  
C. Timchalk ◽  
A. K. Charles
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Activity Ratio ◽  
Cell Membrane Permeability ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Differential Effects ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Differential effects of epigenetic tumor promoters and a genotoxic carcinogen on hepatic cytosolic cyclic adenosine 3′,5′-monophosphate-dependent protein kinase (CAMP-PK) were studied in vitro, since this enzyme is one of the major mediators of cell membrane permeability. Mirex (dodecachlorooctahydro-1,3,4-metheno-2H-cyclobuto[cd]pentalene), like phorbol ester TPA (12-0-tetradecanoylphorbol-13-acetste), caused significant inhibition of cAMP-dependent protein kinase activity ratio, whereas DDT [p, p′-trichlorobis(p-chlorophenyl)ethane] produced concentration-dependent changes. Diethylnitrosamine (DEN) and phenobarbitol (PB), however, showed a significant enhancement of the activity ratio. Interestingly, combinations of mirex, DDT with PB or DEN resulted in the potentiation of CAMP-dependent protein kinase activity in contrast to their effects when used separately. The results suggest that the influences of mirex and TPA in vitro on CAMP-PK are different from those observed in other cell and intact animal systems.

Sign in / Sign up

Export Citation Format

Share Document