The Growth Factor Granulin Interacts with Cyclin T1 and Modulates P-TEFb-Dependent Transcription

Abstract

Cyclin T1, together with the kinase CDK9, is a component of the transcription elongation factor P-TEFb which binds the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. P-TEFb facilitates transcription by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. Cyclin T1 is an exceptionally large cyclin and is therefore a candidate for interactions with regulatory proteins. We identified granulin as a cyclin T1-interacting protein that represses expression from the HIV-1 promoter in transfected cells. The granulins, mitogenic growth factors containing repeats of a cysteine-rich motif, were reported previously to interact with Tat. We show that granulin formed stable complexes in vivo and in vitro with cyclin T1 and Tat. Granulin bound to the histidine-rich domain of cyclin T1, which was recently found to bind to the CTD, but not to cyclin T2. Binding of granulin to P-TEFb inhibited the phosphorylation of a CTD peptide. Granulin expression inhibited Tat transactivation, and tethering experiments showed that this effect was due, at least in part, to a direct action on cyclin T1 in the absence of Tat. In addition, granulin was a substrate for CDK9 but not for the other transcription-related kinases CDK7 and CDK8. Thus, granulin is a cellular protein that interacts with cyclin T1 to inhibit transcription.Human cyclin T1 is a component of positive transcription elongation factor b (P-TEFb) and plays a key role in the activation of human immunodeficiency virus type 1 (HIV-1) transcription by the viral protein Tat (trans-activator of transcription). Cyclin T1 was first isolated as a Tat-binding protein (61) and an orthologue of Drosophila cyclin T (39, 46, 47). P-TEFb contains cyclin T1 and the cyclin-dependent kinase CDK9. This kinase phosphorylates the carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II, thereby facilitating the transition of polymerase II into a productive elongation mode (22, 43, 44, 48-50, 55, 70). The stimulation by Tat of HIV-1 transcriptional elongation and replication is dependent on P-TEFb that contains functional CDK9 and cyclin T1 (9, 11).CDK9 also associates with two additional related cyclins, T2a and T2b, which share their first 642 amino acids. Cyclin T2-CDK9 complexes phosphorylate polymerase II but do not participate in HIV transactivation. The cyclin boxes in the N-terminal regions of cyclins T1 and T2 are 81% identical, while their C-terminal regions are less conserved (47). In spite of this high degree of identity, Tat fails to bind to the T2 cyclins because they lack a crucial cysteine residue at position 261 (14,62). This cysteine is in the Tat-TAR recognition motif of human cyclin T1 that is necessary for its interactions with Tat and TAR, the transactivation response element in the 5Ј untranslated region of all HIV-1 mRNAs (for a review, see reference 26).The activity of the ternary complex P-TEFb-Tat-TAR is modulated by multiple interactions among its components (10,13,22,68). Furthermore, cyclin T1 and CDK9 are present in large...

A Novel Hematopoietic Granulin Induces Proliferation of Goldfish (Carassius auratus L.) Macrophages

“…Progranulin induced the proliferation of embryonic fibroblasts (R-cells) obtained from mice that lack functional insulin-like growth factor-1 receptor (IGF-1). Progranulin was shown to be the only growth factor capable of inducing the proliferation of R-cells in the absence of IGF-1 and platelet-derived growth factor (56), through the activation of the p44/42 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase pathways and induction of cyclin D1 and cyclin B. Interestingly, these pathways are involved in the signaling cascade for IGF-1 and thus may be the reason that progranulin can act in place of IGF-1 (8,57,58). Progranulin was shown to participate in inflammatory responses by inducing cellular migration during wound healing (4,59,60).…”

The granulin gene family: from cancer to dementia

“…Interactions of PGRN with intracellular proteins such as cyclin T have been reported, although their physiological significance is uncertain. (97) GRN knockout and transgenic models Grn knockout mice display behavioral abnormalities but few other recorded phenotypes. (79) Given the biological actions that have been attributed to PGRN, the mild knockout phenotype suggests that PGRN is a molecular generalist, contributing to many tasks but acutely essential for few.…”

Developmental Regulators Containing the I-mfa Domain Interact with T cyclins and Tat and Modulate Transcription

“…This represents another example of zinc ions coordinating interactions between histidine-rich and cysteine-rich domains in separate proteins, as previously shown for granulin, also a cysteine-rich protein that interacts with cyclin T1. 13 The KRM site of cyclins T1 and T2 (Figure 7(d)) includes and extends beyond the TRM, a sequence that is present in human and some other cyclins T1 but not cyclin T2. I-mfa and HIC are the first cellular proteins shown to interact with the TRM, a region that is necessary, but not sufficient, for the binding of HIV-1 Tat and TAR.…”

“…Cells were lysed in 700 μl of IP buffer as previously described. 13 Cell extracts (700 μg protein) were incubated with anti-FLAG M2 affinity gel (Sigma) overnight at 4° C with rocking. Immunocomplexes were washed and analyzed as previously described.…”

“…31,33-44 Their long C termini are divergent but contain a histidine-rich regulatory domain essential for binding RNA polymerase II and other CDK9 substrates and transcription modulators. 13,45,46 Since its discovery, P-TEFb has emerged as an essential factor in diverse cellular systems including cell growth, differentiation and apoptosis. 13,37,39,44,[47][48][49] Another conspicuous role for P-TEFb is in HIV-1 replication 19,30,50 where the viral transcription elongation factor Tat and the viral RNA element TAR (trans-activation response element) associate with P-TEFb complexes that contain cyclin T1.…”