Regulation of Microfilament Organization by Kaposi Sarcoma-associated Herpes Virus-cyclin·CDK6 Phosphorylation of Caldesmon

Abstract

Kaposi sarcoma-associated herpes virus (KSHV) encodes a D-like cyclin (K-cyclin) that is thought to contribute to the viral oncogenicity. K-cyclin activates cellular cyclin-dependent kinases (CDK) 4 and 6, generating enzymes with a substrate selectivity deviant from CDK4 and CDK6 activated by D-type cyclins, suggesting different biochemical and biological functions. Here we report the identification of the actin-and calmodulin-binding protein caldesmon (CALD1) as a novel K-cyclin⅐CDK substrate, which is not phosphorylated by D⅐CDK. CALD1 plays a central role in the regulation of microfilament organization, consequently controlling cell shape, adhesion, cytokinesis and motility. K-cyclin⅐CDK6 specifically phosphorylates four Ser/Thr sites in the human CALD1 carboxyl terminus, abolishing CALD1 binding to its effector protein, actin, and its regulator protein, calmodulin. CALD1 is hyperphosphorylated in cells following K-cyclin expression and in KSHV-transformed lymphoma cells. Moreover, expression of exogenous K-cyclin results in microfilament loss and changes in cell morphology; both effects are reliant on CDK catalysis and can be reversed by the expression of a phosphorylation defective CALD1. Together, these data strongly suggest that K-cyclin expression modulates the activity of caldesmon and through this the microfilament functions in cells. These results establish a novel link between KSHV infection and the regulation of the actin cytoskeleton.Nearly all cellular responses are controlled through protein phosphorylation and the protein kinases catalyzing this process represent the largest single family of enzymes (1). The choice of substrates determines the selectivity of the kinase action and its cellular impact. Therefore, the nature of the substrates of a kinase can provide important clues as to its physiological role and function (2).Recently, several oncogenic gamma herpesviruses have been described to contain within their genome a cyclin-like activator for cyclin-dependent kinases (CDKs) 3 (3). The Kaposi sarcoma herpes virus (KSHV) or human herpes virus 8 (HHV8), a human tumor virus associated with the development of Kaposi sarcoma and several lymphoid malignancies in immunocompromised individuals (4 -6), encodes a cyclin (K-cyclin) that is thought to have descended from cellular D-type cyclins based on co-linearity and sequence identity. Strong evidence from transgenic mouse models suggests that K-cyclin contributes significantly to the oncogenic process elicited by this virus (7,8). D-type cyclins are recognized for their involvement in human oncogenesis (9) and K-cyclin shares their ability to activate the closely related cellular CDK4 and CDK6 and hence phosphorylate and inactivate the retinoblastoma tumor suppressor protein (Rb) (10).In addition to Rb, K-cyclin⅐CDK complexes can phosphorylate proteins that are not substrates for those CDKs when activated by cellular cyclin D. These include the CDK inhibitor p27 KIP , which is normally phosphorylated by cyclin E⅐CDK2, targeting it for degradation b...

Phosphorylation of caldesmon during smooth muscle contraction and cell migration or proliferation

“…Although some of these issues have indeed been clarified for smooth muscle CaD, those concerning non-muscle CaD still remain unresolved after almost two decades. Significantly, the concept of thin filament regulation in non-muscle cells is being supported by quite a few recent reports [7,[9][10][11][12]. Thus it seems timely to revisit this problem now.…”

“…Such phenomena are consistent with the idea that unphosphorylatable l-CaD remains bound to actin filaments even when pertinent kinases are activated, rendering the overly stabilized actin cytoskeleton more difficult to remodel itself. However, like in the case when all seven cdc2 kinase sites were mutated mitosis was only delayed, yet not stopped [5,7], the mutation at Erk sites fails to prevent the cell from eventually changing its shape. Apparently, there are other sites in this region that can be modified by alternative signaling pathways.…”

“…In the smooth muscle system, CaD stabilizes the filamentous organization and inhibits actomyosin interactions [1]. In non-muscle cells CaD is involved in cell migration [2]; it also inhibits Arp2/3 assembly [3,4] and interferes with cell cycle progression [5][6][7]. Binding of CaD to actin is modulated by Ca 2+ /calmodulin (CaM) as well as phosphorylation through kinases such as MAPK, Pak and p34 cdc2 ( Figure 1).…”

Cyclin-Cyclin-dependent Kinase Regulatory Response Is Linked to Substrate Recognition

“…Antibodies-Antibodies used were as follows: rat ␣-K-cyclin (49); PE-␣-CD20 (BD Biosciences); rabbit polyclonal ␣-CDK6 C-21 (Santa Cruz Biotechnology); mouse 9E10 ␣-9E10 (Hybridoma Unit, The Institute of Cancer Research); mouse ␣-p16INK4a50.1 (Santa Cruz Biotechnology); rabbit polyclonal ␣-p27KIP1 C-19 (Santa Cruz Biotechnology); mouse ␣-GAPDH (Advanced ImmunoChemical Inc.); secondary HRP antibodies (Pierce); ␣-hCALD1, ␣-P-hCALD1 730, and ␣-P-hCALD1 789 (40).…”

“…Kinase Assay-Kinase assays were described previously (40). For kinase assays in the presence of CDK inhibitors (GST-p27, GST-p21CIP, and His-p16INK4a) or inhibitory peptides (pRb 866 SNPPKPLKKLRFDIE 880 ; scrambled 15 amino acids KSLNRPFPDKIPELK; E2F1 81 PALGRPPVKRRLDLE 95 (Sigma)), inhibitors were diluted in kinase buffer at the indicated concentrations and incubated with Sf9-produced active kinase complexes for 10 min at room temperature in a final volume of 20 l. Kinase activity was then assayed as above.…”