Brian A. Hemmings

MITOGENIC ACTIVATION, PHOSPHORYLATION, AND NUCLEAR TRANSLOCATION OF PROTEIN KINASE BBETA

Protein kinase B (PKB) is a member of the second messenger-dependent family of serine/threonine kinases that has been implicated in signaling pathways downstream of growth factor receptor tyrosine kinases and phosphatidylinositol 3-kinase. Here we report the characterization of the human β-isoform of PKB (PKBβ). PKBβ is ubiquitously expressed in a number of human tissues, with mRNA and protein levels elevated in heart, liver, skeletal muscle, and kidney. After transfection into HEK-293 or COS-1 cells, PKBβ is activated 2- to 12-fold by mitogens and survival factors. Activation was due to phosphorylation on Thr-309 and Ser-474, which correspond to Thr-308 and Ser-473 implicated in the regulation of PKBα. Both phosphorylation and activation were prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Moreover, membrane-targeted PKBβ was constitutively activated when overexpressed in HEK-293 cells. Although the specific activity of PKBβ was lower than that of PKBα toward Crosstide as a substrate (23 nmol/min/mg compared with 178 nmol/min/mg for PKBα), both enzymes showed similar substrate specificities. Using confocal microscopy, we show that activation of PKBβ results in its nuclear translocation within 20 to 30 min after stimulation. These observations provide evidence that PKBβ undergoes nuclear translocation upon mitogenic activation and support a role for PKB in signaling from receptor tyrosine kinases to the nucleus through phosphatidylinositol 3-kinase.

Reconstitution of a Mg-ATP-dependent protein phosphatase and its activation through a phosphorylation mechanism

A Mg‐ATP‐dependent protein phosphatase has been reconstituted from the catalytic subunit of protein phosphatase‐1 and inhibitor‐2, and consists of a 1:1 complex between these proteins. Activation of this enzyme by glycogen synthase kinase‐3 and Mg‐ATP results from the phosphorylation of inhibitor‐2 on a threonine residue(s) and is accompanied by the dissociation of the complex. The results prove that protein phosphatase‐1 and the Mg‐ATP‐dependent protein phosphatase contain the same catalytic subunit, and that they are interconvertible forms of the same enzyme.

Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Phosphorylation of site 5 by glycogen synthase kinase-5 (casein kinase-II) is a prerequisite for phosphorylation of sites 3 by glycogen synthase kinase-3.

Glycogen synthase kinase‐5 (casein kinase‐II) phosphorylates glycogen synthase on a serine termed site 5. This residue is just C‐terminal to the 3 serines phosphorylated by glycogen synthase kinase‐3, which are critical for the hormonal regulation of glycogen synthase in vivo. Although phosphorylation of site 5 does not affect the catalytic activity, it is demonstrated that this modification is a prerequisite for phosphorylation by glycogen synthase kinase‐3. Since site 5 is almost fully phosphorylated in vivo under all conditions, the role of glycogen synthase kinase‐5 would appear to be a novel one in forming the recognition site for another protein kinase

Identification of the residues on cyclic GMP-dependent protein kinase that are autophosphorylated in the presence of cyclic AMP and cyclic GMP

Autophosphorylation of cyclic GMP-dependent protein kinase (GMP:protein phosphotransferase, EC 2.7.1.37) in the presence of cyclic AMP and Mg-ATP has already been shown to result in the incorporation of up to 2.6 mol phosphate per mol subunit and decrease the A0.5 for cyclic AMP approx. 10-fold. The major sites of autophosphorylation have now been identified as serine-50, threonine-58, serine-72 and threonine-84. Serine-1 and serine-64 are phosphorylated to a minor extent. Threonine-58, which is initially phosphorylated most rapidly, is also the major site that is phosphorylated in the presence of cyclic GMP and Mg-ATP. Since autophosphorylation in the presence of cyclic GMP does not decrease the A0.5 for cyclic AMP, phosphorylation of serine-50, serine-72, or threonine-84 must be responsible for this effect.

Phosphorylation of K-casein by glycogen synthase kinase-3 from rabbit skeletal muscle

Glycogen synthase kinase-3 (ATP:protein phosphotransferase, EC 2.7.1.37) phosphorylated K-casein 20-fold more rapidly than beta-casein, while alpha S1-casein was not a substrate. This distinguished it from casein kinase-I and casein kinase-II, which phosphorylate the beta-casein variant preferentially. Glycogen synthase kinase-3 phosphorylated a serine residue(s) in the C-terminal cyanogen bromide fragment on K-casein. In contrast, cyclic AMP-dependent protein kinase phosphorylated the N-terminal fragment, and phosphorylase kinase the N-terminal and intermediate cyanogen bromide fragments. The results emphasize the potential value of casein phosphorylation as a means of classifying protein kinases.

Classification of Protein Phosphatases Involved in Cellular Regulation

The discovery that glycogen phosphorylase was controlled by a phosphorylation-dephosphorylation mechanism [1] was the first example of enzyme regulation by reversible covalent modification, and phosphorylase kinase [2] and glycogen synthase [3] were the second and third enzymes in which this phenomenon was identified. However, enzyme regulation by phosphorylation-dephosphorylation is not confined to glycogen metabolism and over 20 enzymes are now known to be controlled in this manner [4]. An important generality that seems to be emerging is that enzymes in biodegradative pathways are activated by phosphorylation whereas enzymes in biosynthetic pathways are inactivated by phosphorylation [5, 6] (Table 1). Two implications of this finding are that different enzymes may be regulated by the same protein kinases and protein phosphatases, or that different protein kinases and protein phosphatases may respond to the same effector molecules. These ideas are already established in the case of cyclic AMP-dependent protein kinase (cAMP-PrK) and Ca2+-calmodulin dependent protein kinases.

The Protein Kinase B Family — Structure, Regulation and Function

The phosphorylation of signal transduction molecules is of central importance for growth factor-induced transduction of mitogenic signals, cell growth and differentiation. Stimulation of growth factor-receptor uses by their ligands can activate several signalling modules depending on the cell type. Activation of receptor tyrosine tease cascades can be either independent of a second messenger system or dependent on a second messenger.