CREBtide

cAMP-dependent protein kinase, but not the cGMP-dependent enzyme, rapidly phosphorylates delta-CREB, and a synthetic delta-CREB peptide

Phosphorylation of the cAMP response element binding protein (CREB) by the catalytic subunit of cAMP-dependent protein kinase (cAK) has been implicated in the cAMP-dependent stimulation of gene transcription. delta-CREB, a spliced variant of CREB, and CREBtide (KRREILSRRPSYR), a synthetic peptide based on the phosphorylation sequence in delta-CREB, were tested as substrates of cAK. Phosphorylation of delta-CREB (0.17 microM) was stoichiometric within 30 s when using a concentration of cAK which approximated the intracellular level (0.2 microM). The rate of phosphorylation of delta-CREB was comparable to the rates of the best physiological substrates of cAK tested. The rate of CREBtide phosphorylation was at least as great as that of delta-CREB, indicating that the peptide retained the determinants of delta-CREB which were responsible for substrate efficacy. The apparent Km of CREBtide phosphorylation by cAK was 3.9 microM, which is 10-fold lower than that of kemptide (Km = 39 microM), the synthetic peptide substrate most often employed for cAK measurement. The Vmax values were 12.4 mumol/(min.mg) for CREBtide and 9.8 mumol/(min.mg) for kemptide. The apparent Km of CREBtide phosphorylation by cGMP-dependent protein kinase (cGK) was 2.9 microM and the Vmax value was 3.2 mumol/(min.mg). Both delta-CREB and CREBtide were phosphorylated at a much slower rate by cGK as compared with cAK, implying that the high cAK/cGK specificity exhibited by delta-CREB was retained by the peptide. Taken together, the results indicated that delta-CREB and CREBtide are among the best substrates tested for cAK and suggested that phosphorylation of CREB by this enzyme could occur in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulatory domain determinants that control PKD1 activity

The canonical pathway for protein kinase D1 (PKD1) activation by growth factor receptors involves diacylglycerol binding to the C1 domain and protein kinase C-dependent phosphorylation at the activation loop. PKD1 then autophosphorylates at Ser(916), a modification frequently used as a surrogate marker of PKD1 activity. PKD1 also is cleaved by caspase-3 at a site in the C1-PH interdomain during apoptosis; the functional consequences of this cleavage event remain uncertain. This study shows that PKD1-Δ1-321 (an N-terminal deletion mutant lacking the C1 domain and flanking sequence that models the catalytic fragment that accumulates during apoptosis) and PKD1-CD (the isolated catalytic domain) display high basal Ser(916) autocatalytic activity and robust activity toward CREBtide (a peptide substrate) but little to no activation loop autophosphorylation and no associated activity toward protein substrates, such as cAMP-response element binding protein and cardiac troponin I. In contrast, PKD1-ΔPH (a PH domain deletion mutant) is recovered as a constitutively active enzyme, with high basal autocatalytic activity and high basal activity toward peptide and protein substrates. These results indicate that individual regions in the regulatory domain act in a distinct manner to control PKD1 activity. Finally, cell-based studies show that PKD1-Δ1-321 does not substitute for WT-PKD1 as an in vivo activator of cAMP-response element binding protein and ERK phosphorylation. Proteolytic events that remove the C1 domain (but not the autoinhibitory PH domain) limit maximal PKD1 activity toward physiologically relevant protein substrates and lead to a defect in PKD1-dependent cellular responses.

Cognitive impairment in Coffin-Lowry syndrome correlates with reduced RSK2 activation

Background: Gene expression and protein synthesis, mediated by the transcription factor CREB (cAMP response element binding protein), play an important role in learning and memory in several species, including Drosophila, snails, and mice. Patients with the X-linked disorder Coffin-Lowry syndrome (CLS) have cognitive disabilities, distinctive features, and bony abnormalities as well as mutations in RSK2 (ribosomal S6 kinase-2), a protein kinase that activates CREB by phosphorylation at serine 133. In fibroblasts from a single patient with CLS, epidermal growth factor (EGF)-stimulated CREB phosphorylation was reduced.
Methods: The authors assessed endogenous CREB phosphorylation in a CLS fibroblast line by Western blotting and found impaired CREB phosphorylation in response to stimulation by EGF and the protein kinase C (PKC) agonist phorbol 12-myristate 13-acetate (PMA). They studied RSK2 immunoprecipitated from fibroblasts and lymphoblasts from seven patients with CLS and found a wide range in RSK2's capacity to phosphorylate the synthetic CREB-like peptide, CREBtide, after cell stimulation by PMA.
Results: In lymphoblasts from patients with CLS, PMA-stimulated CREBtide phosphorylation was increased 1.2- to 2.7-fold over baseline, compared to an average fourfold increase in controls. Regression analysis suggested a linear relationship between the magnitude of in vitro RSK2-mediated CREBtide phosphorylation and CLS patient intelligence level (p < 0.05).
Conclusions: This report suggests a correlation between human cognitive performance and cellular capacity to activate RSK2. It provides additional evidence that the CREB kinase, RSK2, and CREB phosphorylation may play important roles in human learning and memory, as they do in lower animals.

A microPLC-based approach for determining kinase-substrate specificity

Phosphorylation is central to signal transduction in living organisms. The specificity of phosphorylation ensures signaling fidelity. Understanding substrate specificity is essential for novel assay development in drug discovery. In this study, we have developed an innovative approach to study protein kinase and its substrate specificity. Using 24 micro parallel liquid chromatography, we studied the reaction kinetics for two different peptide substrates commonly associated with protein kinase A (PKA): Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Glu) and CREBtide (Lys-Arg-Arg-Glu-Ile-Leu-Ser-Arg-Arg-Pro-Ser-Tyr-Arg). The phosphorylation of each substrate was monitored in real time, and the kinetic parameters (V(max), K(m), k(cat), and k(cat) K(m)) were determined for a variety of initial conditions. The results from several kinetic experiments indicated that Kemptide had higher V(max) and k(cat) values compared to CREBtide under the same assay conditions. However, both substrates had a similar k cat)/K(m) value, suggesting that both substrates have similar specificity constants for PKA. We further analyzed the reaction kinetics of ATP for both PKA/substrate complexes. Interestingly, we found that there was a fivefold difference in the specificity constants for ATP affinity to the two complexes, suggesting that even though the sequence differences between the two substrates do not affect their independent interactions with PKA, the differences do have a secondary effect on each enzyme's interaction with ATP and significantly alter the ATP consumption and thus phosphorylation. This novel approach has a broad application for studying enzyme functions and enzyme/substrate specificity.