Phosphotyrosine as a specificity determinant for casein kinase‐2, a growth related Ser/Thr‐specific protein kinase

Abstract

The motif Ser‐Ser‐Ser‐Glu‐Glu is readily phosphorylated by casein kinase‐2 (CK‐2), a growth‐related protein kinase whose consensus sequence is Ser(Thr)‐Xaa‐Xaa‐Glu(Asp) [(1990) Biochim. Biophys. Acta. 1054, 267–283]. Here we show that phosphotyrosine can replace carboxylic acids as specificity determinant for CK‐2 phosphorylation, the phosphotyrosyl peptide Ser‐Ser‐Ser‐TyrP‐TyrP actually being a substrate more efficient than Ser‐Ser‐Ser‐Glu‐Glu itself both in terms of K m (0.69 vs 2.43 mM) and V???. Prior dephosphorylation of phosphotyrosine entirely prevents the subsequent phosphorylation of serine by CK‐2. While Ser‐Ser‐Ser‐TyrP‐TyrP is better than Ser‐Ser‐Ser‐SerP‐SerP, which in turn is better than Ser‐Ser‐Ser‐Glu‐Glu, Ser‐Ser‐Ser‐ThrP‐ThrP is a less efficient substrate than Ser‐Ser‐Ser‐Glu‐Glu. Thus the order of efficiency of phosphoamino acids as specificity determinants for CK‐2 appears to be TyrP>SerP>ThrP.

Phosphorylation of synthetic fragments of inhibitor‐2 of protein phosphatase‐1 by casein kinase‐1 and ‐2

“…< 100 pM) were obtained only using phosphopeptides including one or more phosphoseryl or phosphothreonyl residues on the N-terminal side of the amino acid targeted by CK1, the position -3 being especially effective in this respect (Flotow et al, 1990;Meggio et al, 1991Meggio et al, , 1992. Substitution of phosphorylated with carboxylic side chains (either Glu or Asp), albeit still compatible with fairly high V,,,,, values, invariably caused a rise of K,,, toward the millimolar range (Flotow and Roach, 1991;Meggio et al, 1991 ). It is quite remarkable therefore that the unphosphorylated peptide IDEPSTPYHSMIGDDDDA-YS2"DTETTEA is phosphorylated by CK1 at Ser2O (corresponding to SerX6 of 1-2) with a K,,, value in the low micromolar range (14.3 pM) comparable to that of intact 1-2 (5.3 pM).…”

“…Pertinent to this is also the observation that the pentadecapeptide derived from site Ser174 of the inhibitor-2 peptide 1-2(166-180) displaying the same length and a remarkable similarity with I-2(78-93) (50% identity or conservative substitutions) exhibits a 10-fold higher K,,, (280 vs 33 pM) despite the presence of more N-terminal acidic residues (7 vs 4). It is evident therefore that multiple acidic residues on the N-terminal side, which are necessary in the absence of phosphorylated determinants for site recognition by CK1 (Agostinis et al, 1989;Meggio et al, 1991) are not sufficient alone to ensure highly efficient phosphorylation. It is not clear which o f the additional local features around Ser86 are responsible for its remarkable phosphor-ylation efficiency by CK1.…”

“…Its N-terminal 67 -78 segment including two consecutive predicted P-turns stabilized by Pro70 and Pro73 is not essential since its structural disruption (by replacing the two Pro with Leu) or even its removal in the peptide I-2(78-93), only marginally increases the K,,, values. Also the nature of the carboxylic residues [Asp alone in I-2(78-93) vs Glu and Asp in 12(166-180)] is probably scarcely relevant per se considering that peptides varying in this feature display similar K,,, values (Flotow and Roach, 1991 ;Meggio et al, 1991). A priori it was possible that the high phosphorylation efficiency of the peptide I-2(78-93) as opposed to I-2(166-180) could be determined by its C-terminal moiety which is quite divergent from that of the latter peptide.…”

“…The superiority of such phosphopeptides was mostly accounted for by their low K, values, 10-100-fold below those of their derivatives bearing carboxylic, instead of phosphorylated, residues. These latter invariably display K,,, values in the millimolar range (Agostinis et al, 1989;Flotow et al, 1990;Meggio et al, 1991).…”

“…This concept rests mostly on experiments with synthetic peptide substrates whose efficient phosphorylation by CK1 is crucially dependent on the presence of phosphate groups at position -3 and, less effectively, -4, relative to the target serine (Flotow et al, 1990;Meggio et al, 1991). This is not the case with 1-2 whose efficient targetting by CK1 neither requires previous phosphorylation by other protein kinase(s) nor is impaired by previous treatment with protein phosphatases (Agostinis et al, 1987).…”