Molecular basis for the substrate specificity of protein kinase B; comparison with MAPKAP kinase‐1 and p70 S6 kinase

Abstract

The substrate specificity of protein kinase-Bcc (PKB~ also known as RAC kinase or Akt) was investigated using synthetic peptide substrates related to the sequence surrounding the phosphorylation site on glycogen synthase kinase-3 (GSK3). The minimum sequence motif required for efficient phosphorylation was Arg-Xaa-Arg-Yaa-Zaa-Ser/ThrHyd, where Xaa is any amino acid, Yaa and Zaa are small residues other than glycine and Hyd is a bulky hydrophobic residue (Phe, Leu). The most effective substrate, Arg-Pro-ArgThr-Ser-Ser-Phe, was phosphorylated with a Km of 5 ttM and ma~ of 260 U/rag. PKBcx phosphorylated histone H2B (Km 5 ~NI, Vmax 68 U/rag) specifically at Ser-36 which also lies in an Arg-Xaa-Arg-Xaa-Xaa-Ser-Hyd motif. The peptide Arg-ProArg-Ala-Ala-Thr-Phe may be a relatively specific substrate for PKBcx because, unlike other substrates, it is not phosphorylated by p70 $6 kinase or MAP kinase activated protein (MAPKAP) kinase-1. . All forms of PKB possess an Nt;rminal pleckstrin homology (PH) domain, followed by a ttalytic domain and a short C-terminal tail. The catalytic c omain is most similar to cyclic AMP-dependent protein kixase (PKA, 65% similarity) and to protein kinase C (PKC, "5% similarity), findings that gave rise to two of its names, x amely PKB (i.e. between PKA and PKC) and RAC (Related 1 ) A and C kinase).PKBc~ is activated within 1 min following stimulation of tells with insulin [8,9]. The activation of PKBc~ by insulin , r IGF-1 is accompanied by its phosphorylation at Thr-308 and Ser-473, and phosphorylation of both of these residues is required to achieve a high level of activity [10]. Like the activation of PKB~ [11,12], the phosphorylation of Thr-308 and Ser-473 is prevented by wortmannin, an inhibitor of phosphoinositide (PI) 3-kinase [10].The only physiological substrate for PKBc~ to have been identified to date is the protein kinase glycogen synthase kinase-3 (GSK3). GSK3 is inhibited in response to insulin with a half-time of 2 min, slightly slower than that for activation of PKBcx (1 min) and inhibition results from its phosphorylation at the same serine residue (Ser-21 in GSK3~ and Ser-9 in GSK3fi) which is targetted by PKB~x in vitro. Like the activation of PKBc~, the inhibition of GSK3 by insulin is prevented by the PI 3-kinase inhibitors wortmannin and LY 294002.GSK3cz and GSK3[3 are phosphorylated at Ser-21 and Ser-9, respectively, by two other insulin-stimulated protein kinases, namely p70 $6 kinase and MAP kinase-activated protein kinase-I (MAPKAP-KI, also known as p90 $6 kinase). However, these enzymes are not rate-limiting for the inhibition of GSK3 by insulin in L6 myotubes because specific inhibitors of their activation (rapamycin-p70 $6 kinase; PD 98059-MAPKAP kinase-1) have no effect [9]. The inhibition of GSK3 is thought to contribute to the stimulation of glycogen synthesis [9] and protein synthesis [13] by insulin.The activation of PI 3-kinase is essential for many of the effects of insulin and growth factors, including the stimulation of glucose transport, fatty...

EGF induced SOS phosphorylation in PC12 cells involves P90 RSK-2

“…The two putative Rsk consensus sites within this stretch are boxed in lighter grey and the targeted serine residue shown in bold type. The serine residues identi®ed by Corbalan-Garcia et al (1996b) (Kohn et al, 1995;Marte et al, 1996), but it diplays a phosphorylation consensus sequence reminiscent of that of p90 Rsk-2 (R-X-R-Y-Z-S-Hyd, where X is any amino acid, Y and Z are small amino acids other than glycine and Hyd is a bulky hydrophobic amino acid) (Alessi et al, 1996). However, Akt is unlikely to be a kinase involved in SOS phosphorylation and down regulation since the puri®ed kinase was unable to phosphorylate the two di erent Gst fusions corresponding to the carboxyl terminal region of SOS (data not shown).…”

The emerging role of the PI3-K-Akt pathway in prostate cancer progression

“…Phosphorylated Akt (P-Akt) then dissociates from the plasma membrane and translocates to nucleus and other subcellular compartments to phosphorylate and regulate the function of many cellular proteins involved in cell proliferation, survival, motility and angiogenesis processes that are critical for tumorigenesis and metastasis. 3,5,9,10 The primary consequence of PI3-K activation is the generation of phospholipids at the membrane, which function as second messengers to activate downstream kinases, including PDK1 and ILK. It is not clear whether ILK is an immediate upstream kinase of Akt.…”

Genetic interactions underlying otic placode induction and formation

“…These proteins become sequestered to the cytoplasm when phosphorylated, preventing their activity as transcriptional regulators (Biggs et al, 1999;Brunet et al, 1999;Brownawell et al, 2001). Foxi1 contains a protein kinase B (PKB) phosphorylation consensus sequence similar to that of the Fox O class of proteins (unpublished observations; Alessi et al, 1996) and, thus, may be regulated in a similar manner, perhaps in response to FGF signals. The results presented here do not distinguish between these possibilities and other similar mechanisms.…”