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 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 Phosphorylation of the inhibitory subunit of troponin in perfused hearts of mice deficient in phosphorylase kinase. Evidence for the phosphorylation of troponin by adenosine 3′:5′-phosphate-dependent protein kinase in vivo

1977 ◽  
Vol 168 (2) ◽  
pp. 307-310 ◽  
Author(s):  
P J England
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Troponin I ◽  
Protein Kinase Activity ◽  
Phosphorylase Kinase ◽  
Dependent Protein Kinase ◽  
Parallel Increase ◽  
Dependent Protein

When hearts from control and phosphorylase kinase-deficient (I strain) mice were perfused with 0.1 micrometer-DL-isoprenaline, there was a parallel increase in contraction, cyclic AMP concentration and troponin I phosphorylation. However, there was no increase in phosphorylase a in the I-strain hearts, whereas the control hearts showed a large increase. Assays of I-strain heart extracts showed a normal cyclic AMP-dependent protein kinase activity but no phosphorylase kinase activity. It is concluded that troponin I is phosphorylated in intact hearts by protein kinase and not phosphorylase kinase.

scholarly journals Proteolytic Cleavage of the Catalytic Subunit of DNA-Dependent Protein Kinase during Poliovirus Infection

2004 ◽  
Vol 78 (12) ◽  
pp. 6313-6321 ◽  
Author(s):  
Kareem L. Graham ◽  
Kurt E. Gustin ◽  
Carlos Rivera ◽  
N. Muge Kuyumcu-Martinez ◽  
Sunny S. Choe ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Caspase 3 ◽  
Proteolytic Cleavage ◽  
Catalytic Subunit ◽  
Dependent Protein Kinase ◽  
Poliovirus Infection ◽  
Dependent Protein ◽  
2A Protease

ABSTRACT DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that has critical roles in DNA double-strand break repair, as well as B- and T-cell antigen receptor rearrangement. The DNA-PK enzyme consists of the Ku regulatory subunit and a 450-kDa catalytic subunit termed DNA-PKCS. Both of these subunits are autoantigens associated with connective tissue diseases such as systemic lupus erythematosus (SLE) and scleroderma. In this report, we show that DNA-PKCS is cleaved during poliovirus infection of HeLa cells. Cleavage was visible as early as 1.5 h postinfection (hpi) and resulted in an approximately 40% reduction in the levels of native protein by 5.5 hpi. Consistent with this observation, the activity of the DNA-PKCS enzyme was also reduced during viral infection, as determined by immunoprecipitation kinase assays. Although it has previously been shown that DNA-PKCS is a substrate of caspase-3 in vitro, the protein was still cleaved during poliovirus infection of the caspase-3-deficient MCF-7 cell line. Cleavage was not prevented by infection in the presence of a soluble caspase inhibitor, suggesting that cleavage in vivo was independent of host caspase activation. DNA-PKCS is directly cleaved by a picornaviral 2A protease in vitro, producing a fragment similar in size to the cleavage product observed in vivo. Taken together, our results indicate that DNA-PKCS is cleaved by the 2A protease during poliovirus infection. Proteolytic cleavage of DNA-PKCS during poliovirus infection may contribute to inhibition of host immune responses. Furthermore, cleavage of autoantigens by viral proteases may target these proteins for the autoimmune response by generating novel, or “immunocryptic,” protein fragments.

scholarly journals Autophosphorylation of the Catalytic Subunit of the DNA-Dependent Protein Kinase Is Required for Efficient End Processing during DNA Double-Strand Break Repair

2003 ◽  
Vol 23 (16) ◽  
pp. 5836-5848 ◽  
Author(s):  
Qi Ding ◽  
Yeturu V. R. Reddy ◽  
Wei Wang ◽  
Timothy Woods ◽  
Pauline Douglas ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Strand Break ◽  
Catalytic Subunit ◽  
Dna Breaks ◽  
End Joining ◽  
Dependent Protein Kinase ◽  
Major Cluster ◽  
Dependent Protein

ABSTRACT The DNA-dependent protein kinase (DNA-PK) plays an essential role in nonhomologous DNA end joining (NHEJ) by initially recognizing and binding to DNA breaks. We have shown that in vitro, purified DNA-PK undergoes autophosphorylation, resulting in loss of activity and disassembly of the kinase complex. Thus, we have suggested that autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) may be critical for subsequent steps in DNA repair. Recently, we defined seven autophosphorylation sites within DNA-PKcs. Six of these are tightly clustered within 38 residues of the 4,127-residue protein. Here, we show that while phosphorylation at any single site within the major cluster is not critical for DNA-PK's function in vivo, mutation of several sites abolishes the ability of DNA-PK to function in NHEJ. This is not due to general defects in DNA-PK activity, as studies of the mutant protein indicate that its kinase activity and ability to form a complex with DNA-bound Ku remain largely unchanged. However, analysis of rare coding joints and ends demonstrates that nucleolytic end processing is dramatically reduced in joints mediated by the mutant DNA-PKcs. We therefore suggest that autophosphorylation within the major cluster mediates a conformational change in the DNA-PK complex that is critical for DNA end processing. However, autophosphorylation at these sites may not be sufficient for kinase disassembly.

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