Determinants for substrate phosphorylation by p21-activated protein kinase (γ-PAK)

Polygena T. Tuazon, William Spanos, Edwin L. Gump, Curtis A. Monnig, Jolinda A. Traugh

Research output: Contribution to journalArticle

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Abstract

γ-PAK, originally designated PAK I and subsequently identified as a member of the p21-activated protein kinase family, has been shown to have cytostatic properties and to be involved in maintaining cells in a nondividing state [Rooney, R. D., et al., (1996) J. Biol. Chem. 271, 21498- 21504]. The determinants for phosphorylation of substrates by γ-PAK have been identified by examining the kinetics of phosphorylation of a series of synthetic peptides patterned after the sequence KKRKSGL, which is the site phosphorylated by γ-PAK in the Rous sarcoma virus nucleocapsid protein NC in vivo and in vitro. With these peptides, the recognition sequence for γ-PAK has been shown to contain two basic amino acids in the -2 and -3 positions, as represented by (K/R)RXS, in which the -2 position is an arginine the -3 position is an arginine or a lysine, and X can be an acidic, basic, or neutral amino acid. A basic amino acid in the -1 or -4 position improves the rate of phosphorylation by increasing the V(max) and decreasing the K(m). An acidic amino acid in the -1 position increases the rate (2.5-fold), as does an acidic residue in the -4 position, although to a lower extent (1.6-fold). Proline in the -1 or +1 position has a deleterious effect and inhibits phosphorylation by γ-PAK. The substrate requirements of protein kinases that recognize basic amino acids on the N-terminal side of the phosphorylatable residue such as cAMp-dependent protein kinase (PKA) and Ca2+/phospholipid- dependent protein kinase (PKC) have been compared with γ-PAK using the same peptides. An acidic residue in the -1 position negatively affects PKA and PKC; thus, peptides containing the sequence KRES can be used to identify γ- PAK.

Original languageEnglish (US)
Pages (from-to)16059-16064
Number of pages6
JournalBiochemistry
Volume36
Issue number51
DOIs
StatePublished - Dec 1 1997

Fingerprint

p21-Activated Kinases
Phosphorylation
Basic Amino Acids
Protein Kinases
Acidic Amino Acids
Substrates
Peptides
Arginine
Neutral Amino Acids
Nucleocapsid Proteins
Rous sarcoma virus
Cytostatic Agents
Viruses
Proline
Lysine
Phospholipids
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Determinants for substrate phosphorylation by p21-activated protein kinase (γ-PAK). / Tuazon, Polygena T.; Spanos, William; Gump, Edwin L.; Monnig, Curtis A.; Traugh, Jolinda A.

In: Biochemistry, Vol. 36, No. 51, 01.12.1997, p. 16059-16064.

Research output: Contribution to journalArticle

Tuazon, Polygena T. ; Spanos, William ; Gump, Edwin L. ; Monnig, Curtis A. ; Traugh, Jolinda A. / Determinants for substrate phosphorylation by p21-activated protein kinase (γ-PAK). In: Biochemistry. 1997 ; Vol. 36, No. 51. pp. 16059-16064.
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AB - γ-PAK, originally designated PAK I and subsequently identified as a member of the p21-activated protein kinase family, has been shown to have cytostatic properties and to be involved in maintaining cells in a nondividing state [Rooney, R. D., et al., (1996) J. Biol. Chem. 271, 21498- 21504]. The determinants for phosphorylation of substrates by γ-PAK have been identified by examining the kinetics of phosphorylation of a series of synthetic peptides patterned after the sequence KKRKSGL, which is the site phosphorylated by γ-PAK in the Rous sarcoma virus nucleocapsid protein NC in vivo and in vitro. With these peptides, the recognition sequence for γ-PAK has been shown to contain two basic amino acids in the -2 and -3 positions, as represented by (K/R)RXS, in which the -2 position is an arginine the -3 position is an arginine or a lysine, and X can be an acidic, basic, or neutral amino acid. A basic amino acid in the -1 or -4 position improves the rate of phosphorylation by increasing the V(max) and decreasing the K(m). An acidic amino acid in the -1 position increases the rate (2.5-fold), as does an acidic residue in the -4 position, although to a lower extent (1.6-fold). Proline in the -1 or +1 position has a deleterious effect and inhibits phosphorylation by γ-PAK. The substrate requirements of protein kinases that recognize basic amino acids on the N-terminal side of the phosphorylatable residue such as cAMp-dependent protein kinase (PKA) and Ca2+/phospholipid- dependent protein kinase (PKC) have been compared with γ-PAK using the same peptides. An acidic residue in the -1 position negatively affects PKA and PKC; thus, peptides containing the sequence KRES can be used to identify γ- PAK.

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