Abstract
Synthetic peptides have been used to define the consensus amino acid sequence for substrate recognition by the meiosis-activated myelin basic protein (MBP) kinase (p44mpk), which was purified from maturing sea star oocytes. This protein kinase shares many properties with the mitogen-activated microtubule-associated protein-2 kinase (p42mapk) in vertebrates. Recently, Thr-97 in the tryptic fragment KNIVTPRTPPPSQGK of bovine MBP was identified as the major site of phosphorylation by p44mpk (Sanghera, J. S., Aebersold, R., Morrison, H. D., Bures, E. J., and Pelech, S. L. (1990) FEBS Lett. 273, 223-226). Synthetic peptides modeled after this sequence revealed that the presence of a proline residue C-terminal (+1 position) to the phosphorylatable threonine (or serine) residue was critical for recognition by p44mpk. Although not essential, a proline residue located at the -2 position enhanced the Vmax of peptide phosphorylation. Basic, acidic, and non-polar residues were equally tolerated at the -1 position. The presence of an amino acid residue at position -3 also increased peptide phosphorylation. Thus, the optimum consensus sequence for phosphorylation by p44mpk was defined as Pro-X-(Ser/Thr)-Pro, where X is a variable amino acid residue, but ideally not a Pro. Peptides that included this sequence were phosphorylated by p44mpk with Vmax values approaching 1 mumol.min-1.mg-1 and with apparent Km values of approximately 1 mM). Pseudosubstrate peptides in which the phosphorylatable residue was replaced by valine or alanine were weak inhibitors of p44mpk (apparent Ki values of approximately 3 mM). Over 40 distinct protein kinases contain Pro-X-(Ser/Thr)-Pro sequences including the human receptors for insulin and epidermal growth factor, and kinases encoded by the human proto-oncogenes abl, neu, and raf-1, and Schizosaccharomyces pombe cell cycle control genes ran-1 and wee-1. Multiple putative sites were also identified in rat microtubule-associated protein-2, human retinoblastoma protein, human tau protein, and Drosophila myb protein and RNA polymerase II.
Highlights
Synthetic peptides ulated in concertwith the Myelin basic protein (MBP) kinases in maturing seastar modeled after this sequence revealed that the presen(c1e)and Xenopus laeuis oocytes (2,4,5).~ 4 4 ' i"s i~tse~lf subject of a proline residue C-terminal (+1 position) to the to activation by protein-serine and protein-tyrosinephosphosphorylatablethreonineresiduewas phorylation during sea star oocytes maturation, the critical for recognitionby ~ 4 4 " A~ l~tho. ugh not essen- responsible protein kinases have yet to be identified (6)
Tial, a proline residue located at the -2 position en- The physical and enzymatic properties of ~ 4 4 " a' r~e ~rehancedthe VmaXof peptidephosphorylation.Basic, markably similar to those of a murine 42-kDa mitogen-actiacidic, and non-polar residues were tolerated vated protein kinase ( ~ 4 2 " " ~th~a)t is activated during the Go at the -1 position
Of phosphorylated bovine MBP permitted the identification of Thr-97inthe sequence KNIVTPRTPPPSQGKasthe major site of phosphorylation of this protein by ~ 4 4 ' ("10~).~
Summary
Definition of the Optimum Consensus Sequence for Substrate Recognitionby p44 "'pk-Automated solid-phase Edman sequencing of a tryptic fragment (containingresidues 91-104). Once it was established that the Thr residue at the fifth position in the S1 peptide (Thr-94 in MBP) was not required for phosphorylation, latter peptides were synthesized as amides for reasons of economy. Fur- and K , values were obtained when the basic Arg residue at thermore, the placement of a Pro at the-1 position, as in the position -1 in the S5 peptide was converted either to a S11 peptide, resulted in a -24-fold decrease in the rate of nonpolar residue (for example, Ala (S14 peptide), Leu (S15 phosphorylation by ~ 4 4 " 'r~ela~tive to that of the S5 peptide peptide), or Phe (S16 peptide))or the polar residue Gln Converted to anAla (Sl2peptide), the V,,, of phosphorylation Some protein-Ser/Thr kinases are known to exhibita was decreased greater than 20-fold and the apparentK,,, was marked preference for the phosphorylatable amino acid residue.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
