Determination of the Plk4/Sak consensus phosphorylation motif using peptide spots arrays

Genie C. Leung; Cynthia S.W. Ho; Ivan M. Blasutig; James M. Murphy; Frank Sicheri

doi:10.1016/j.febslet.2006.11.080

Determination of the Plk4/Sak consensus phosphorylation motif using peptide spots arrays

Genie C. Leung
, Cynthia S.W. Ho
, Ivan M. Blasutig
, James M. Murphy
, Frank Sicheri

Research output: Contribution to journal › Article › Research › peer-review

23 Citations (Scopus)

Abstract

The family of polo like kinases (Plks) regulate cell cycle progression through key functional roles in mitosis. While the four mammalian family members, Plk1-4, share overlapping functions, each member possesses unique functions that may be dictated in part by their ability to phosphorylate different substrates. Numerous cellular substrates for Plk1, 2, and 3 have been characterized, but the protein targets for Plk4/Sak remain unknown. We have purified the kinase domain of Sak and demonstrated that it has robust kinase activity in vitro. Using in vitro kinase assays on peptide spots arrays, we determined the consensus phosphorylation motif for Sak to be ¥-[Ile/Leu/Val]-Ser/Thr-φ{symbol}-φ{symbol}-X-¥/Pro (where φ{symbol} denotes a large hydrophobic residue, ¥ is a charged residue dependent on the context of the surrounding sequence, and residues in brackets are unfavoured). This consensus phosphorylation motif differs from that of Plk1, and provides a basis for future studies to identify in vivo substrates of Sak.

Original language	English
Pages (from-to)	77-83
Number of pages	7
Journal	FEBS Letters
Volume	581
Issue number	1
DOIs	https://doi.org/10.1016/j.febslet.2006.11.080
Publication status	Published - 9 Jan 2007
Externally published	Yes

Keywords

Consensus phosphorylation motif
Peptide arrays
Plk4/Sak
Polo like kinases
Protein kinase specificity

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10.1016/j.febslet.2006.11.080

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title = "Determination of the Plk4/Sak consensus phosphorylation motif using peptide spots arrays",

abstract = "The family of polo like kinases (Plks) regulate cell cycle progression through key functional roles in mitosis. While the four mammalian family members, Plk1-4, share overlapping functions, each member possesses unique functions that may be dictated in part by their ability to phosphorylate different substrates. Numerous cellular substrates for Plk1, 2, and 3 have been characterized, but the protein targets for Plk4/Sak remain unknown. We have purified the kinase domain of Sak and demonstrated that it has robust kinase activity in vitro. Using in vitro kinase assays on peptide spots arrays, we determined the consensus phosphorylation motif for Sak to be ¥-[Ile/Leu/Val]-Ser/Thr-φ\{symbol\}-φ\{symbol\}-X-¥/Pro (where φ\{symbol\} denotes a large hydrophobic residue, ¥ is a charged residue dependent on the context of the surrounding sequence, and residues in brackets are unfavoured). This consensus phosphorylation motif differs from that of Plk1, and provides a basis for future studies to identify in vivo substrates of Sak.",

keywords = "Consensus phosphorylation motif, Peptide arrays, Plk4/Sak, Polo like kinases, Protein kinase specificity",

author = "Leung, \{Genie C.\} and Ho, \{Cynthia S.W.\} and Blasutig, \{Ivan M.\} and Murphy, \{James M.\} and Frank Sicheri",

note = "Funding Information: We thank D. Briant, N. Warner, and B. Seet for helpful discussion and assistance with the Multipep peptide spots array synthesis, to P. Metalnikov and his mass spectrometry lab for their help in defining the Sak KIM domain boundaries, and to T. Pawson for the use of his Multipep peptide synthesizer. We also thank M. Tyers and T. Pawson for the full length human ASP, APC1, and yeast MSC3 constructs. This work was supported by grants from the National Cancer Institute of Canada and the Canadian Institutes of Health Research to F.S., and from the Terry Fox Foundation through an award from the National Cancer Institute of Canada to G.C.L. G.C.L. is a recipient of a Bank of Montreal Fellowship in Medical Research. J.M.M. is a recipient of a C.J. Martin Fellowship from the National Health and Medical Research Council of Australia. ",

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doi = "10.1016/j.febslet.2006.11.080",

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T1 - Determination of the Plk4/Sak consensus phosphorylation motif using peptide spots arrays

AU - Leung, Genie C.

AU - Ho, Cynthia S.W.

AU - Blasutig, Ivan M.

AU - Murphy, James M.

AU - Sicheri, Frank

N1 - Funding Information: We thank D. Briant, N. Warner, and B. Seet for helpful discussion and assistance with the Multipep peptide spots array synthesis, to P. Metalnikov and his mass spectrometry lab for their help in defining the Sak KIM domain boundaries, and to T. Pawson for the use of his Multipep peptide synthesizer. We also thank M. Tyers and T. Pawson for the full length human ASP, APC1, and yeast MSC3 constructs. This work was supported by grants from the National Cancer Institute of Canada and the Canadian Institutes of Health Research to F.S., and from the Terry Fox Foundation through an award from the National Cancer Institute of Canada to G.C.L. G.C.L. is a recipient of a Bank of Montreal Fellowship in Medical Research. J.M.M. is a recipient of a C.J. Martin Fellowship from the National Health and Medical Research Council of Australia.

PY - 2007/1/9

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N2 - The family of polo like kinases (Plks) regulate cell cycle progression through key functional roles in mitosis. While the four mammalian family members, Plk1-4, share overlapping functions, each member possesses unique functions that may be dictated in part by their ability to phosphorylate different substrates. Numerous cellular substrates for Plk1, 2, and 3 have been characterized, but the protein targets for Plk4/Sak remain unknown. We have purified the kinase domain of Sak and demonstrated that it has robust kinase activity in vitro. Using in vitro kinase assays on peptide spots arrays, we determined the consensus phosphorylation motif for Sak to be ¥-[Ile/Leu/Val]-Ser/Thr-φ{symbol}-φ{symbol}-X-¥/Pro (where φ{symbol} denotes a large hydrophobic residue, ¥ is a charged residue dependent on the context of the surrounding sequence, and residues in brackets are unfavoured). This consensus phosphorylation motif differs from that of Plk1, and provides a basis for future studies to identify in vivo substrates of Sak.

AB - The family of polo like kinases (Plks) regulate cell cycle progression through key functional roles in mitosis. While the four mammalian family members, Plk1-4, share overlapping functions, each member possesses unique functions that may be dictated in part by their ability to phosphorylate different substrates. Numerous cellular substrates for Plk1, 2, and 3 have been characterized, but the protein targets for Plk4/Sak remain unknown. We have purified the kinase domain of Sak and demonstrated that it has robust kinase activity in vitro. Using in vitro kinase assays on peptide spots arrays, we determined the consensus phosphorylation motif for Sak to be ¥-[Ile/Leu/Val]-Ser/Thr-φ{symbol}-φ{symbol}-X-¥/Pro (where φ{symbol} denotes a large hydrophobic residue, ¥ is a charged residue dependent on the context of the surrounding sequence, and residues in brackets are unfavoured). This consensus phosphorylation motif differs from that of Plk1, and provides a basis for future studies to identify in vivo substrates of Sak.

KW - Consensus phosphorylation motif

KW - Peptide arrays

KW - Plk4/Sak

KW - Polo like kinases

KW - Protein kinase specificity

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U2 - 10.1016/j.febslet.2006.11.080

DO - 10.1016/j.febslet.2006.11.080

M3 - Article

C2 - 17174311

AN - SCOPUS:33845660449

SN - 0014-5793

VL - 581

SP - 77

EP - 83

JO - FEBS Letters

JF - FEBS Letters

IS - 1

ER -

Determination of the Plk4/Sak consensus phosphorylation motif using peptide spots arrays

Abstract

Keywords

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