Characterization of Synthetic Peptide Substrates for p34<sup>cdc2</sup> Protein Kinase

Marshak, Daniel R.; Vandenberg, Mark; Bae, Young‐Seuk; Yu, Ii Je

doi:10.1002/jcb.240450413

Cited by 43 publications

(45 citation statements)

References 41 publications

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“…HeLa cells were grown in suspension as previously described (27). HeLa were incubated together at 30°C for 12 min prior to the addition of K52R.…”

Section: Methodsmentioning

confidence: 99%

“…Affinity-purified antibodies were isolated on an MKK1 peptide column and shown to be specific for MKK1 by immunoblotting and immunoprecipitating baculovirus-expressed MKK1 (data not shown). Antiserum specific for p34cdc (G6) was generated in rabbits a ainst the peptide carboxy terminus (11) 5); it corresponded to the p34cdc2-associated p62 cyclin that is phosphorylated by p34cdc2 (2,11,27).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation.

Rossomando¹,

Dent²,

Sturgill³

et al. 1994

Mol. Cell. Biol.

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Mitogen-activated protein kinase kinase 1 (MKK1), a dual-specificity tyrosine/threonine protein kinase, has been shown to be phosphorylated and activated by the raf oncogene product as part of the mitogen-activated protein kinase cascade. Here we report the phosphorylation and inactivation of MKK1 by phosphorylation on threonine 286 and threonine 292. MKK1 contains a consensus phosphorylation site for p34cdc2, a serine/ threonine protein kinase that regulates the cell division cycle, at Thr-286 and a related site at Thr-292. p34cdc2 catalyzes the in vitro phosphorylation of MKK1 on both of these threonine residues and inactivates MKK1 enzymatic activity. Both sites are phosphorylated in vivo as well. The data presented in this report provide evidence that MKK1 is negatively regulated by threonine phosphorylation.The regulation of mammalian cell growth is accomplished by the integration of extracellular signals, such as polypeptide growth factors, with intracellular phosphorylation cascades extending from the plasma membrane to the cell nucleus. Recent studies have indicated that mitogen-activated protein (MAP) kinases (33, 41), also known as extracellular signalregulated kinases (7), are central to these intracellular signal transduction pathways. Two MAP kinases, p42maPk and p44mapk, have been well characterized in many mammalian cell systems (33,41). Both enzymes are phosphorylated on tyrosine and threonine and are activated by a novel protein kinase termed MAP kinase kinase (MKK), also known as MAP kinase/extracellular signal-regulated kinase (MEK) kinase (9,15,29,34,37). The activity of MKK is specific for the regulatory residues, threonine 183 and tyrosine 185, in p42maPk (32), suggesting that MKK is a key regulator of MAP kinases in the cell.The regulation of the MKK/MAP kinase pathway is evolving as a network of crosstalking protein kinases and phosphatases that exert both positive and negative signals. For example, the cellular proto-oncogene product, c-Raf-1, catalyzes the phosphorylation and activation of MKK (10,11,23). Furthermore, the effects of other upstream protein kinases on MKK activity are also becoming apparent. Several groups have demonstrated a link between the MKK pathway and cyclic AMPdependent protein kinase (3,8,17,39,43 protein kinase activity (9,15,29,34,36). Although this method of inactivation has not been documented in vivo, observations suggest that additional phosphorylation(s) of MKK occurs following its initial activation and leads to increased susceptibility to inactivation by phosphatase 2A in vitro (1). Taken together, these observations demonstrate that both protein kinases and phosphatases act upon MKK directly or indirectly after its initial phosphorylation and activation to rigorously control the activity of the MKK/MAP kinase pathway.Recently, two isoforms of MKK, known as MKK1 and MKK2, were identified, and their amino acid sequences were elucidated (9,31,38,45). MKK1 contains a consensus sequence for phosphorylation by p34cdc2, a key regulator of the cell division cyc...

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“…HeLa cells were grown in suspension as previously described (27). HeLa were incubated together at 30°C for 12 min prior to the addition of K52R.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation.

Rossomando¹,

Dent²,

Sturgill³

et al. 1994

Mol. Cell. Biol.

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“…act. 912 nmoles/min per mgl purified from mitotic HeLa cells and characterized according to Marshak et al ( 1991 ) …”

Section: Phosphorylation Of P53mentioning

confidence: 99%

A proteolytic fragment from the central region of p53 has marked sequence-specific DNA-binding activity when generated from wild-type but not from oncogenic mutant p53 protein.

Bargonetti¹,

Manfredi²,

Chen³

et al. 1993

Genes & Development

262

181

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; 2Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 USA p53 is a sequence-specific DNA-binding oligomeric protein that can activate transcription from promoters bearing p53-binding sites. Whereas the activation region of p53 has been identified within the amino terminus, the location of the specific DNA-binding domain has not been reported. Thermolysin treatment of p53 protein generates a stable protease-resistant fragment that binds with marked specificity to p53 DNA-binding sites. Amino-terminal sequencing of the fragment located the thermolysin cleavage site to residue 91. Because the fragment does not contain the cdc2 phosphorylation site at Ser-315, we conclude that the the site-specific DNA-binding domain of p53 spans the central region of the protein. The vast majority of the mutations in oncogenically derived p53 proteins are located within this central portion of the molecule. Such mutant p53 proteins exhibit defective sequence-specific DNA-binding. Although thermolysin digestion of mutant p53 proteins generates proteolytic patterns that differ from wild-type protein, one mutant tested, His-273, generates a resistant fragment that migrates with a similar electrophoretic mobility to the wild-type protease-resistant fragment. Interestingly, although intact mutant His-273 protein binds to DNA at 20~ the thermolysin-resistant mutant fragment does not. In addition, the central protease-resistant, site-specific binding region of wild-type p53 does not demonstrate nonspecific DNA-binding. Thus, although sequences outside of the central region of p53 contribute to both nonspecific DNA-binding and oligomerization, they are not required for sequence-specific DNA-binding.

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“…p34cdc2 was purified from nuclear extracts from nocodazole-treated HeLa cells (61). This preparation exhibited no detectable contamination with seven other classes of protein kinases, including PKC, cyclic AMP-dependent protein kinase (PKA), casein kinase II (CKII), glycogen synthase kinase III (GSK-3), phosphorylase kinase, and calmodulin kinase II.…”

mentioning

confidence: 99%

Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts.

Baker¹,

Kerppola²,

Luk³

et al. 1992

Mol. Cell. Biol.

126

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c-jun is a member of the family of immediate-early genes whose expression is induced by factors such as serum stimulation, phorbol ester, and differentiation signals. Here we show that increased Jun synthesis after serum stimulation is accompanied by a concomitant increase in phosphorylation. Several serine-threonine kinases were evaluated for their ability to phosphorylate Jun in vitro. p34cdc2, protein kinase C, casein kinase II, and pp44mapk phosphorylated Jun efficiently, whereas cyclic AMP-dependent protein kinase and glycogen synthase kinase III did not. The sites phosphorylated by p34cdc2 were similar to those phosphorylated in vivo after serum induction. The major sites of phosphorylation were mapped to serines 63, 73, and 246. Phosphorylation of full-length Jun with several kinases did not affect the DNA-binding activity of Jun homodimers or Fos-Jun heterodimers. Comparison of the DNA binding and in vitro transcription properties of wild-type and mutated proteins containing either alanine or aspartic acid residues in place of Ser-63, -73, and -246 revealed only minor differences among homodimeric complexes and no differences among Fos-Jun heterodimers. Thus, phosphorylation of Jun did not produce a significant change in dimerization, DNA-binding, or in vitro transcription activity. The regulatory role of phosphorylation in the modulation of Jun function is likely to be considerably more complex than previously suggested.

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Characterization of Synthetic Peptide Substrates for p34^cdc2 Protein Kinase

Cited by 43 publications

References 41 publications

Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation.

Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation.

A proteolytic fragment from the central region of p53 has marked sequence-specific DNA-binding activity when generated from wild-type but not from oncogenic mutant p53 protein.

Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts.

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Characterization of Synthetic Peptide Substrates for p34cdc2 Protein Kinase

Cited by 43 publications

References 41 publications

Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation.

Mitogen-activated protein kinase kinase 1 (MKK1) is negatively regulated by threonine phosphorylation.

A proteolytic fragment from the central region of p53 has marked sequence-specific DNA-binding activity when generated from wild-type but not from oncogenic mutant p53 protein.

Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts.

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Characterization of Synthetic Peptide Substrates for p34^cdc2 Protein Kinase