Polygena T. Tuazon

[33] Casein kinase I

Publisher Summary Casein kinase I is a multipotential, cyclic nucleotide-independent, Ca 2+ independent protein kinase, which phosphorylates acidic substrates including casein. The type I casein kinases is identified and described in a variety of eukaryotes including yeast, plants, fowl, and mammals. The enzyme is present in nuclei and cytoplasm and found associated with membranes, ribosomes, and mitochondria. The type I casein kinases are termed “multipotential enzymes” because they phosphorylate a number of different substrates. The type I casein kinase is distinct from both the type II casein kinases, which are also present in many eukaryotic tissues, and the mammary gland enzymes, which function in the physiological phosphorylation of casein. Although casein kinase I has been shown to modify a number of different substrates, the functional role for the enzyme in metabolism remains to be elucidated. It is postulated that the casein kinases function to integrate metabolism because of the large number and variety of substrates identified for the enzymes.

Multisite autophosphorylation of p21-activated protein kinase gamma-PAK as a function of activation.

p21-activated protein kinase (PAK) is a family of serine/threonine kinases whose activity is stimulated by binding to small G-proteins such as Cdc42 and subsequent autophosphorylation. Focusing on the ubiquitous γ-isoform of PAK in this study, baculovirus-infected insect cells were used to obtain recombinant γ-PAK, while native γ-PAK was isolated from rabbit reticulocytes. Two-dimensional gel electrophoresis of γ-PAK followed by immunoblot analysis revealed a similar profile for native and recombinant γ-PAK, both consisting of multiple protein spots. Following Cdc42-stimulated autophosphorylation, the two-dimensional profiles of native and recombinant γ-PAK were characterized by a similar acidic shift, suggesting a common response to Cdc42. To understand the effect of differential phosphorylation on its activation status, γ-PAK autophosphorylation was conducted in the presence or absence of activators such as Cdc42 and histone II-AS, followed by tryptic digestion and comparative two-dimensional phosphopeptide mapping. The major phosphopeptides were subjected to a combination of manual and automated amino acid sequencing. Overall, eight autophosphorylation sites were identified in Cdc42-activated γ-PAK, six of which are in common with those previously reported in α-PAK, while Ser-19 and Ser-165 appear to be uniquely phosphorylated in the γ-form. Further, the phosphorylation of Ser-141, Ser-165, and Thr-402 was found to correlate with γ-PAK activation.

p21-activated protein kinase γ-PAK in pituitary secretory granules phosphorylates prolactin

p21‐activated protein kinase γ‐PAK phosphorylates prolactin (PRL) in rat pituitary secretory granules on Ser‐177 and on the equivalent site, Ser‐179, in recombinant human PRL. This is shown by comparison of phosphopeptide maps with the human PRL mutant S179D. γ‐PAK is present in rat and bovine granules as identified by in‐gel phosphorylation of histone H4, and by immunoblotting. Thus, phosphorylation of PRL by γ‐PAK in granules produces the PRL molecule that has been shown to antagonize the growth‐promoting activity of unmodified PRL, and is consistent with the identified role of γ‐PAK in the induction and maintenance of cytostasis.

Cdc42-independent activation and translocation of the cytostatic p21-activated protein kinase γ-PAK by sphingosine

Autophosphorylation of p21‐activated protein kinase γ‐PAK is stimulated at 10 μM sphingosine in vitro and is maximal at 100 μM. Sites autophosphorylated on γ‐PAK in response to sphingosine are identical to those obtained with Cdc42(GTP). Autophosphorylation is paralleled by stimulation of γ‐PAK activity as measured with peptide and protein substrates. In 3T3‐L1 cells, sphingosine stimulates the autophosphorylation and activity of γ‐PAK associated with the membrane‐containing particulate fraction by 2.8‐fold, but does not stimulate the activity of the soluble enzyme. Thus, γ‐PAK is activatable via a Cdc42‐independent mechanism, suggesting sphingosine has a role in γ‐PAK activation under conditions of cell stress.

p21-Activated kinase 2 (PAK2) inhibits TGF-β signaling in Madin-Darby canine kidney (MDCK) epithelial cells by interfering with the receptor-Smad interaction.

Background: PAK2 is a mediator of TGF-β in mesenchymal cells. However, whether PAK2 could modulate TGF-β signaling remains elusive. Results: PAK2 associates with Smad2/3 and phosphorylates Smad2 at Ser417, thus inhibiting the TGF-β-induced Smad2-TβRI association and signal transduction in MDCK cells. Conclusion: PAK2 inhibits TGF-β signaling in MDCK epithelial cells. Significance: This study unravels a novel regulatory mechanism of R-Smad activity. TGF-β (transforming growth factor β) plays a variety of cellular functions mainly through the Smad pathway. Phosphorylation of the carboxyl SXS motif in R-Smads (Smad2 and Smad3) by the type I receptor TβRI is a key step for their activation. It has been reported that the serine/threonine kinase PAK2 (p21-activated kinase 2) can mediate TGF-β signaling in mesenchymal cells. Here, we report that PAK2 restricts TGF-β-induced Smad2/3 activation and transcriptional responsiveness in MDCK epithelial cells. Mechanistically, PAK2 associates with Smad2 and Smad3 in a kinase activity-dependent manner and blocks their activation. PAK2 phosphorylates Smad2 at Ser-417, which is adjacent to the L3 loop that contributes to the TβRI-R-Smad association. Consistently, substitution of Ser-417 with glutamic acid attenuates the interaction of Smad2 with TβRI. Together, our results indicate that PAK2 negatively modulate TGF-β signaling by attenuating the receptor-Smad interaction and thus Smad activation.

Phosphatidylinositol kinases from rabbit reticulocytes

Phosphatidylinositol (PI) kinase was isolated from the postribosomal supernatant of rabbit reticulocytes. This activity was identified by the formation of a product that comigrated with phosphatidylinositol-4-phosphate (PIP) when purified PI was phosphorylated in the presence of (/sup 32/P)ATP and Mg/sup 2 +/. Three major peaks of PI kinase activity were resolved by chromatography on DEAE-cellulose. The first peak eluted at 50-100 mM NaCl together with several serine protein kinases, casein kinase (CK) I and protease activated kinase (PAK) I and II. The PI kinase was subsequently separated from the protein kinases by chromatography on phosphocellulose. The second peak eluted at 125-160 mM NaCl and contained another lipid kinase activity that produced a product which comigrated with phosphatidic acid on thin layer chromatography. The third peak, which eluted at 165-200 mM NaCl, partly comigrated with casein kinase (CK) II and an active protein kinase(s) which phosphorylated mixed histone and histone I. CK II and the histone kinase activities were also separated by chromatography on phosphocelluslose. The different forms of PI kinase were characterized and compared with respect to substrate and salt requirements.

Phosphorylation of myosin light chain by protease activated kinase I.

Abstract Protease activated kinase I from rabbit reticulocytes has been shown to phosphorylate the P-light chain of myosin light chains isolated from rabbit skeletal muscle. The enzyme is not activated by Ca 2+ and calmodulin or phospholipids. Protease activated kinase I is not inhibited by trifluoperazine at concentrations up to 200 μM or by the antibody to the Ca 2+ , calmodulin-dependent myosin light chain kinase from rabbit skeletal muscle. Two-dimensional peptide mapping of chymotryptic digests of myosin P-light chain show the site phosphorylated by the protease activated kinase is different from that phosphorylated by the Ca 2+ , calmodulin-dependent myosin light chain kinase.

A MEMBRANE-BOUND PROTEIN KINASE FROM RABBIT RETICULOCYTES IS AN ACTIVE FORM OF MULTIPOTENTIAL S6 KINASE

An active ribosomal protein S6 kinase has been highly purified from the membranes of rabbit reticulocytes by chromatography of the Triton X-100 extract on DEAE-cellulose, SP-Sepharose Fast Flow, and by FPLC on Mono Q and Superose-12. The S6 kinase elutes around 40 000 daltons upon gel filtration on Superose-12 or Sephacryl S-200. It has a subunit molecular weight of 40-43 kDa as determined by protein kinase activity following denaturation/renaturation in SDS-polyacrylamide gels containing S6 peptide. It also phosphorylates translational initiation factors eIF-2 and eIF-4F, glycogen synthase, histone 1, histone 2B, myelin basic protein, but not prolactin, skeletal myosin light chain, histone 4, tubulin, and casein. Apparent Km values have been determined to be 15 microM for ATP, 1.2 microM for S6 and 10 microM for S6 peptide. Two-dimensional tryptic phosphopeptide mapping shows the same sites on S6 are phosphorylated as those identified previously with proteolytically activated multipotential S6 kinase from rabbit reticulocytes, previously denoted as protease activated kinase II. Examination of relative rates of phosphorylation and kinetic constants of synthetic peptides based on previously identified phosphorylation sites, indicates a minimum substrate recognition sequence to be arginine at the n - 3 position. Based on these characteristics, including molecular weight and an expanded substrate specificity, the membrane S6 kinase can be distinguished from the p90 (Type I) and p70 (Type II) S6 kinases, and from protein kinase C and the catalytic subunit of cAMP-dependent protein kinase.

CYCLIC NUCLEOTIDE-INDEPENDENT PROTEIN KINASES FROM RABBIT RETICULOCYTES AND PHOSPHORYLATION OF TRANSLATIONAL COMPONENTS

Five different cyclic nucleotide-independent protein kinases have been isolated from rabbit reticulocytes. The enzymes are distinct from the type I and type II cAMP-dependent protein kinases and the free catalytic subunit of these enzymes. Three of the cyclic nucleotide-independent protein kinases, casein kinase I, casein kinase II and the hemin controlled repressor, have been obtained in highly purified form. The physical and chemical properties of these enzymes have been studied and the possible modes of regulation examined. In addition, two protease-activated kinases have also been observed in reticulocytes. These five cyclic nucleotide-independent protein kinases have different substrate specificities with respect to histone and casein. All of the protein kinases, cyclic nucleotide-independent and cAMP-dependent, differentially phosphorylate components of the protein synthesizing system including 40S ribosomal subunits and initiation factors 2, 3, 4B and 5. Each of the protein kinases phosphorylates two or more of these components and the initiation factors and ribosomal subunits are in turn modified by at least two different protein kinases. The result is a multiply phosphorylated system. Alterations in the phosphorylation state of these components can be observed under conditions which activate or inhibit specific protein kinase activities.