Insulin Receptor Substrate 1 Binds Two Novel Splice Variants of the Regulatory Subunit of Phosphatidylinositol 3-Kinase in Muscle and Brain

Abstract

We have identified two novel alternatively spliced forms of the p85␣ regulatory subunit of phosphatidylinositol (PI) 3-kinase by expression screening of a human skeletal muscle library with phosphorylated baculovirus-produced human insulin receptor substrate 1. One form is identical to p85␣ throughout the region which encodes both Src homology 2 (SH2) domains and the inter-SH2 domain/p110 binding region but diverges in sequence from p85␣ on the 5 side of nucleotide 953, where the entire break point cluster gene and SH3 regions are replaced by a unique 34-amino-acid N terminus. This form has an estimated molecular mass of ϳ53 kDa and has been termed p85/AS53. The second form is identical to p85 and p85/AS53 except for a 24-nucleotide insert between the SH2 domains that results in a replacement of aspartic acid 605 with nine amino acids, adding two potential serine phosphorylation sites in the vicinity of the known serine autophosphorylation site (Ser-608). Northern (RNA) analyses reveal a wide tissue distribution of p85␣, whereas p85/AS53 is dominant in skeletal muscle and brain, and the insert isoforms are restricted to cardiac muscle and skeletal muscle. Western blot (immunoblot) analyses using an anti-p85 polyclonal antibody and a specific anti-p85/AS53 antibody confirmed the tissue distribution of p85/AS53 protein and indicate a ϳ7-fold higher expression of p85/AS53 protein than of p85 in skeletal muscle. Both p85 and p85/AS53 bind to p110 in coprecipitation experiments, but p85␣ itself appears to have preferential binding to insulin receptor substrate 1 following insulin stimulation. These data indicate that the gene for the p85␣ regulatory subunit of PI 3-kinase can undergo tissue-specific alternative splicing. Two novel splice variants of the regulatory subunit of PI 3-kinase are present in skeletal muscle, cardiac muscle, and brain; these variants may have important functional differences in activity and may play a role in tissue-specific signals such as insulin-stimulated glucose transport or control of neurotransmitter secretion or action.Insulin binding of the insulin receptor leads to the tyrosine phosphorylation of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) and Shc, which can then interact with and modulate the activity of a number of proteins via Src homology 2 (SH2) domains, including phosphatidylinositol (PI) 3-kinase, the protein tyrosine phosphatase SHPTP-2, and Grb2, which, acting through Sos can stimulate Ras GTPase activity (reviewed in references 6, 36, and 37). Recent studies have indicated an important role for PI 3-kinase, especially in insulin stimulation of glucose transport (18,20,22,28,39), p70 S6 kinase (20, 28), glycogen synthesis (27), and antilipolysis (22). PI 3-kinase is a heterodimer composed of an 85-kDa regulatory subunit (p85␣) and a 110-kDa catalytic subunit (p110␣). Isoforms of p85␣ and p110␣ have been identified and termed p85␤ (23) and p110␤. In addition, a G-protein-activated form of PI 3-kinase, termed p110␥, which is not dependent on the 85-kDa regulatory s...

Lower Phosphoinositide 3-Kinase (PI 3-kinase) Activity and Differential Expression Levels of Selective Catalytic and Regulatory PI 3-Kinase Subunit Isoforms in Prefrontal Cortex and Hippocampus of Suicide Subjects

“…In addition, we observed that changes in PI 3-kinase were present in all suicide subjects whether they were diagnosed with major depression or other psychiatric illnesses, which further suggests that these changes are related to suicide rather than some specific psychopathology. A number of studies have shown that all catalytic and regulatory isoforms of class IA PI 3-kinase, except p110d, are highly expressed in the central nervous system (Pons et al, 1995;Antonetti et al, 1996;Fruman et al, 1996;Inukai et al, 1997). When we examined the relative mRNA distribution of catalytic and regulatory subunit isoforms of PI 3-kinase, an interesting pattern of expression emerged in the human PFC and hippocampus.…”

Class IA phosphatidylinositol 3-kinase regulates osteoclastic bone resorption through protein kinase B–mediated vesicle transport

“…(40) Mice lacking only p85a are viable, (41) but mice lacking p85a, p55a, and p50a die immediately after birth, (34) indicating that p55a and p50a can compensate for the loss of p85a in vivo. In addition, PI3K activity is enhanced in mice lacking only p85a compared with wild-type mice, possibly because of higher p50a/p55a expression.…”

Phosphoinositide 3-kinases as a common platform for multi-hormone signaling

“…Pik3r1 can be expressed in three splice variants that encode p85a, p55a, and p50a respectively; on the other hand, Pik3r2 and Pik3r3 are known to present only one transcript and encode p85b and p55g respectively. While p85a and p85b are ubiquitously expressed (Gout et al 1992), p50a and p55a are present in fat, muscle, liver, and brain (Antonetti et al 1996, Inukai et al 1996, and p55g is mainly expressed in the brain (Pons et al 1995). All members of the p85 family contain a p110-binding region tethering them to a specific domain located at the N-terminal end of class IA p110s.…”