Internal amino acid sequence analysis of the 80 kDa protein kinase C substrate from rat brain: Relationship to the 87 kDa substrate from bovine brain

Abstract

We have obtained sequence data from five proteolytic peptides totalling 102 amino acid residues of the 80 kDa protein kinase C substrate purified from rat brain. The amino acid sequences of these five peptides were compared with that deduced from a cDNA encoding the 87 kDa protein kinase C substrate from bovine brain. The overall amino acid sequence identity within the regions covered by our peptides is 54%. Two peptides aligned at the C-and N-termini of the bovine protein kinase C substrate with a very high degree of homology (more than 80% identity). Two other peptides exhibited 62% and 46% identity with two regions located in the C-terminal half of the bovine protein. The fifth peptide which contains the sequence PEQPEQPEQ did not reveal any similarity with the bovine protein. Based on the homologies of our experimentally determined sequences, which represent about 30% of the deduced sequence of the bovine protein, we suggest that although these protein kinase C substrates are not identical, they may belong to a family of related proteins.

Purification and internal amino acid sequence of the 80 kDa protein kinase C substrate from Swiss 3T3 fibroblasts

“…Previous studies have shown that the 80 kDa PKC substrate from Swiss 3T3 fibroblasts is heat stable [ 18,191, Therefore, a purification scheme involving heat treatment, DEAE-cellulose chromatography, G-l 50 gel filtration and electroelution from preparative SDS polyacrylamide gels was followed as previously described for the purification of the rat brain protein [19,24]. Approximately 10 lug of homogeneous 80 kDa protein was isolated from the cytosolic fraction of 2.3 x 10" confluent, quiescent Swiss 3T3 fibroblasts (Table I).…”

“…2. The amino acid sequences of peptides A-E1 of the Swiss 3T3 fibroblast 80 kDa protein were aligned with the deduced amino acid sequence of the bovine brain 87 kDa PKC substrate [23] and with proteolpic peptides from the rat brain 80 kDa PKC substrate [24] (unpublished data). Vertical lines between amino acids denote identical residues.…”

“…After electrophoresis, protein was visualized by brief staining with Coomassie blue (0.1070). The band corresponding to the 80 kDa protein was excised and electroeluted from the gel slice as previously described [24]. A summary of the purification of the 80 kDa protein from 2.3 x 10" Swiss 3T3 fibroblasts is shown in Table I. 2.2.5.…”

“…The purification and sequencing of proteolytic peptides of the rat brain 80 kDa PKC substrate enabled a comparison to be made of the rat and bovine proteins. This revealed an overall homology of 54% for the regions covered by the peptides [24]. Hence, these acidic PKC substrates are similar but not identical.…”

“…Indeed, there has been a recent report on the cloning and sequencing of an acidic 80 kDa PKC substrate from the human epidermal carcinoma cell line A431 [27]. Comparison of the deduced amino acid se-quence of this protein with peptides generated from the rat brain 80 kDa PKC substrate [24] or the deduced amino acid sequence of the 87 kDa bovine brain protein 1231, indicates that the protein from A431 cells is unrelated to the acidic 80-87 kDa PKC substrates isolated from brain. Consequently, it was important to determine whether the 80 kDa protein detected in nonexcitable Swiss 3T3 fibroblasts, which are a model cell line for elucidating mitogenic signalling pathways, expresses an 80 kDa PKC substrate related to any of those identified in brain or cultured epidermal carcinoma cells.…”