MARCKS phosphorylation by individual protein kinase C isozymes in insect Sf9 cells.

Relatively little is known about the substrate specificity of individual protein kinase C (PKC) isozymes, particularly with respect to physiologically relevant substrates. One class of prominent cellular substrates for PKC is represented by the myristoylated alanine-rich C kinase substrate, or MARCKS, protein. In the present study, we have used a baculovirus expression system to coexpress human MARCKS with eight different isozymes of PKC, to determine which isozymes are capable of phosphorylating MARCKS in intact cells. In Sf9 cells, coexpression of MARCKS with individual PKC isozymes led to the following increases in MARCKS phosphorylation: alpha, 3.6-fold; beta iota, 4.6-fold; beta mu, 2.7-fold; gamma, 4.8-fold; delta, 3.0-fold; epsilon, 4.3-fold; and eta, 4.9-fold. In most cases, stimulation of cells with a phorbol ester led to a slight increase (20-30%) in MARCKS phosphorylation. PKC zeta did not phosphorylate MARCKS to any appreciable extent above control. In addition, in vitro kinetic analysis of PKC zeta showed that it has a 1000-fold lower affinity for a synthetic peptide comprising the MARCKS phosphorylation site domain compared to mixed conventional PKC isozymes from rat brain. These data indicate that MARCKS is a substrate in intact cells for at least seven isozymes of PKC: alpha; beta iota; beta mu; gamma; delta; epsilon; and eta. The isozyme PKC zeta does not appear to phosphorylate MARCKS in vivo or with significant affinity in vitro. Thus, PKC zeta, which is not activated by phorbol esters or diacylglycerol, also appears to behave differently with respect to this class of important cellular PKC substrates.

The production of an active protein kinase C-delta in insect cells is greatly enhanced by the use of the basic protein promoter.

Occasionally, only a small percentage of recombinant proteins produced in the baculovirus expression system are functionally active. We had previously shown that the majority of protein kinase C-delta (PKC-delta) produced in insect cells was inactive; less than 1% of the expressed enzyme had lipid-dependent kinase activity. In this report, we have attempted to optimize the production of a catalytically active PKC-delta. Under optimum conditions, we were able to increase the levels of PKC-delta from 10-20% to about 65% of the total cellular protein; however, there was no increase in the levels of catalytically active enzyme. Expression of PKC-delta as a fusion protein or as a secreted protein also met with limited success. Under all conditions, expression of PKC-delta proteins under control of the strong polyhedrin promoter resulted in the production of large amounts of inactive enzyme. Expression under the control of the basic protein promoter, Pcor, resulted in the reduction of the levels of recombinant protein by a factor of about four, but the PKC-delta enzyme produced under these conditions was 10- to 15-fold more active. Thus, the earlier temporal expression of PKC-delta in insect cells resulted in the production of more active enzyme.

A comparison of large-scale Sf9 insect cell growth and protein production: stirred vessel vs. airlift.

A simple and relatively inexpensive stirred-vessel system for the large-scale growth of insect cells (Sf9) is described. Sf9 cell growth in a stirred-vessel fermentor and an airlift fermentor were compared on the basis of maximum cell density and average population doubling time. Also, both fermentor systems were compared with respect to the large-scale production of a recombinant human protein (protein kinase C-eta). No significant differences in Sf9 cell growth or protein expression levels were apparent between the two fermentor systems. However, large differences in cost and scale-up of each system are discussed with respect to the large-scale production of recombinant proteins.

Molecular and biochemical characterization of a recombinant human PKC-delta family member.

Two cDNA clones coding for the human protein kinase C-delta (PKC-delta) were fortuitously isolated during the process of screening a human library for a cDNA clone of an unrelated protein, the nucleolar protein fibrillarin. The two human homologues have about 88% nucleotide sequence identity to the rat and mouse PKC-delta cDNA clones. A comparison of the predicted amino acid sequences of the two human PKC-delta clones with the rat and mouse homologues indicated a greater degree of sequence divergence (89-90% homology) compared to the high degree of sequence conservation observed with other human PKC family members and their mammalian counterparts. Expression of the clones in the baculovirus insect-cell expression system indicated that both proteins exhibited phorbol ester binding activity, and were dependent upon phosphatidylserine and diacylglycerol for maximal activation. Further characterization of the properties of the human PKC-delta revealed substrate and lipid dependencies distinct from other members of the protein kinase C family; including PKC-deltas isolated from other species. The dissimilarities in the predicted amino acid sequences between the human and other mammalian species could account in part for some of these observed biochemical differences.